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REVIEW

Functional links between telomeres and proteins of the DNA-damage response

Fabrizio d’Adda di Fagagna,1,3,4 Soo-Hwang Teo,2,3 and Stephen P. Jackson2,5

1IFOM Foundation—The FIRC Institute of Molecular Oncology Foundation, 20139 Milan, Italy; 2The Wellcome Trust and Cancer Research UK Gurdon Institute, and Department of Zoology, University of Cambridge, Cambridge CB2 1QR, UK

In response to DNA damage, cells engage a complex set important roles in regulating normal telomeric func- of events that together comprise the DNA-damage re- tions. In this review, we focus on the role of DDR factors sponse (DDR). These events bring about the repair of the in regulating telomere length and stability, and also ex- damage and also slow down or halt cell cycle progression plain how dysfunctional telomeres can trigger the DDR. until the damage has been removed. In stark contrast, Before doing this, however, we first summarize the sa- the ends of linear chromosomes, telomeres, are generally lient features of both telomeres and the DDR. not perceived as DNA damage by the cell even though they terminate the DNA double-helix. Nevertheless, it Telomere structure and biology has become clear over the past few years that many pro- The ends of linear chromosomes contain long stretches teins involved in the DDR, particularly those involved in of DNA tandem repeats (TTAGGG in vertebrates) and responding to DNA double-strand breaks, also play key terminate in a 3Ј protruding single-stranded DNA over- roles in telomere maintenance. In this review, we dis- hang. Due to the inability of the standard lagging-strand cuss the current knowledge of both the telomere and the DNA replication machinery to copy the most distal telo- DDR, and then propose an integrated model for the mere sequences (i.e., those at the very end of the chro- events associated with the metabolism of DNA ends in mosome) and to the additional exonucleolytic processing these two distinct physiological contexts. needed to generate protruding overhangs at both ends, telomeric DNA progressively decreases in length as cells All organisms respond to interruptions in the DNA go through successive division cycles. Hence, in the ab- double-helix by promptly launching the DNA-damage sence of specialized telomere homeostatic mechanisms response (DDR). This involves the mobilization of DNA- this would ultimately lead to the loss of all telomeric repair factors and the activation of pathways, often sequences and subsequently to the loss of more internal termed checkpoint pathways, which temporarily or per- essential genetic information and ensuing cell death. To manently delay cell cycle progression. Although the in- circumvent this, many cells maintain their telomeres by tegrity of the DNA double-helix is perturbed by telo- the action of telomerase, a specialized reverse transcrip- meres (the ends of linear chromosomes), these structures tase that uses its associated RNA component as a tem- generally escape activating the DDR. Several explana- plate to elongate the TG-rich telomeric DNA strand. Al- tions have been proposed to explain the exceptional na- though in vitro telomerase activity is dependent on the ture of telomeres in this regard. Thus, it has been sug- activity of the reverse transcriptase catalytic subunit gested that a telomere might not be recognized by com- (Est2p in the budding yeast Saccharomyces cerevisiae; ponents of the DDR because of its unique DNA TERT in mammals) and the telomerase RNA template sequence and structure, its specific localization within (Tlc1 in S. cerevisiae and hTR in humans), other factors the cell nucleus, and/or because of the actions of specific are clearly needed for telomerase action in vivo (see proteins associated with it. Although this is partly cor- Table 1). For instance, effective telomerase function in S. rect, recent findings have revealed that, contrary to ini- cerevisiae requires Est1p and Est3p, and the loss of either tial expectations, various proteins involved in the DDR of these two proteins—like the loss of Tlc1 or Est2p— physically associate with telomeres and actually play leads to progressive telomere shortening (for review, see Blackburn 2000). Furthermore, and as explained below, effective telomerase action in vivo also requires several [Keywords: Telomere; DNA-damage response; checkpoint; senescence; proteins associated with the DDR. DNA repair] The telomeric repeat sequences are essential for many 3These authors contributed equally to this work. Corresponding authors: of the key biological features of telomeres by virtue of 4E-MAIL [email protected]; FAX 39-02-574303-231. them being recognized by a specific set of sequence- and 5E-MAIL [email protected]; FAX 44-1223-334089. Article and publication are at http://www.genesdev.org/cgi/doi/10.1101/ structure-specific DNA-binding factors (Table 1; Fig. 1). gad.1214504. Some of these bind to the double-stranded portion of the

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d’Adda di Fagagna et al.

Table 1. Telomere-associated factors

Mammals S. cerevisiae S. pombe C. elegans

TRF1: telomere DNA binder Rap1p: telomere length Taz1p: telomere length and and telomerase mediated- regulator structure regulator telomere length regulator Tbf1p: telomere binding factor TIN2: telomerase mediated telomere length regulator TANK1: TRF1 PARP modifier and telomere length regulator TANK2: TRF1 PARP modifier TRF2: telomere DNA binder with telomere end capping function and telomerase independent telomere length regulator RAP1: TRF2 interactor and Rap1p: Taz1p interactor and telomere length regulator telomere length regulator ERCC1/XPF: TRF2 interacting endonuclease MRN complex: TRF2 interactor MRX complex: telomere length MRN: in vivo component of and single stranded overhang the telomere and telomere regulator length regulator Rif1: Trf2 interactor and in Rif1/2p: Rap1p interactors and Rif1p: Taz1p interactor and vivo component of mouse telomere length regulators telomere length regulator telomeres POT1: TRF1 interactor and Cdc13p: single stranded Pot1p: single stranded single stranded telomeric telomeric DNA binder with telomeric DNA binder DNA binder with telomere telomere capping and with telomere capping length regulation functions telomerase recruiting functions functions Stn1p/Ten1p: Cdc13p interactors and mediators of its telomerase recruiting and capping functions Ku: in vivo component of the Ku: in vivo component of the Ku: in vivo component of telomere and telomere length telomere, telomere length the telomere and telomere regulator (?) and single-stranded overhang length regulator regulator DNA-PKcs: in vivo component of the telomere with telomere capping functions EST1A/B: telomere length Est1p: in vivo cofactor of Est1p: in vivo cofactor of regulator (?) telomerase telomerase TERT: catalytic component of Est2p: catalytic component of Trt1p: catalytic component the telomerase complex the telomerase complex of the telomerase complex TR: RNA component of the Tlc1: RNA component of the telomerase complex telomerase complex PARP1: telomere length regulator RPA: in vivo component of the telomere with Est1p-recruiting functions 9–1–1 complex: in vivo MRT-2 and Hus1: regulators component of the of telomere length and telomere and telomere germline mortality length regulator Tel2p: telomere length Rad5: telomere length regulator regulator (?) telomeric DNA and are involved in telomere length while others have important roles in capping the very regulation (e.g. S. cerevisiae Rap1p, Schizosaccharomy- end of the chromosome by virtue of their ability to rec- ces pombe Taz1p, and mammalian TRF1 and TRF2), ognize the telomeric 3Ј overhang (e.g., S. cerevisiae

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Telomeres and the DNA-damage response

Figure 1. Schematic representation of telomere factors in different organisms.

Cdc13, S. pombe Pot1p, and possibly hPOT1). These lat- associated with it, or a combination of both, that is cru- ter factors bind single-stranded DNA through a con- cial for evading the activation of a DDR. In vitro, the served OB (oligonucleotide/oligosaccharide binding) fold mammalian telomere repeat binding protein, TRF2, can domain (Mitton-Fry et al. 2002; Lei et al. 2003) and be- promote T-loop formation (Stansel et al. 2001), and im- cause a DDR ensues in their absence, are believed to play pairing the DNA-binding function of TRF2 in vivo leads crucial roles in preventing the inappropriate triggering of to either ataxia telangiectasia mutated (ATM)- and p53- the DDR by the telomere. Indeed, in S. cerevisiae lacking dependent cell death or to permanent cell cycle arrest, functional Cdc13p, the CA-rich telomeric strand depending on the cell type (Karlseder et al. 1999). Al- complementary to that bound by Cdc13p is rapidly de- though T loops have so far only been demonstrated in graded, leading to RAD9-dependent cell-cycle arrest mammals and Trypanosomes (Munoz-Jordan et al. 2001), (Garvik et al. 1995; see below). Similarly, inactivation of similar structures may exist in other organisms. In S. S. pombe Pot1p leads to rapid and dramatic telomere cerevisiae, evidence has been provided that telomeric shortening, leaving chromosome circularization as the DNA can loop back in a manner that requires Sir3p, a preferred option to maintain cell viability (Baumann and protein needed for the formation of transcriptionally si- Cech 2000). lent chromatin flanking telomeres and at certain other In addition to it being bound by the proteins described genomic loci (de Bruin et al. 2001). However, it should be above, there is evidence that telomeric DNA may adopt noted that inactivation of SIR proteins does not in itself an unusual and specific structure, the so-called T loop. In trigger a DDR, indicating that if yeast does fold its telo- this structure, the very end of the chromosome is folded meric termini by SIR-dependent mechanisms, it must back and the single-stranded telomeric 3Ј overhang is also employ other systems to prevent telomeres from tucked into a portion of the double-stranded telomeric normally being recognized as DNA damage. These re- DNA, resulting in a three-stranded structure (Griffith et sults also suggest that T loops and telomere chromatin al. 1999). This conformation has been suggested to pre- looping-back in yeasts represent functionally different vent telomere ends from being recognized as DNA dam- structures. age and triggering the DDR. Nevertheless, it is still un- Another feature of telomeres in some eukaryotic cells clear whether it is the structure per se or the factors is that at various cell cycle stages they appear to cluster

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Figure 2. Schematic hierarchical representation of the factors activated following the generation of DNA damage.

and position preferentially at the nuclear periphery sume. In multi-cellular organisms an inability to repair (Scherthan 2001). Although this is mostly associated DNA damage and/or prolonged checkpoint activation with chromosomal separation during meiosis, at least in can also lead to programmed cell death (apoptosis; Rich S. cerevisiae and Plasmodium falciparum it also occurs et al. 2000), or cause the cell to enter into permanent cell during interphase of the mitotic cell cycle (Gotta et al. cycle arrest—a state known as senescence (Schmitt 1996; Figueiredo et al. 2002). In such situations, the telo- 2003). Other aspects of the DDR include changes in meres form clusters in perinuclear chromatin domains chromatin structure at sites of DNA damage (Fernandez- that constitute areas of transcriptional repression and Capetillo and Nussenzweig 2004) and the transcriptional modulate recombination between internal tracts of yeast induction and posttranslational modification of DNA- telomeric DNA (Stavenhagen and Zakian 1998; Figueir- repair and checkpoint proteins as well as other proteins edo et al. 2002). In S. cerevisiae, the telomeres appear to that indirectly influence DNA repair, for example by be tethered to such locations in part via their interaction modulating deoxyribonucleotide availability (Zhou and with the DNA repair protein Ku (see below). To date, Elledge 2000; Rouse and Jackson 2002a). however, there is no firm evidence for analogous mecha- Although it is often useful to study specific aspects of nisms operating in other eukaryotes. the DDR in isolation, recent findings have suggested that these distinctions are somewhat arbitrary. For ex- ample, in some situations “DNA-repair” factors are The DNA-damage response needed to process initial DNA lesions into structures The DDR has evolved to optimize cell survival following that can trigger checkpoint activation, and “checkpoint DNA damage and control the proliferation of a damaged proteins” can control the activity of DNA-repair factors cell. Probably the best characterized—and most highly and their recruitment to sites of DNA damage (Lydall evolutionarily conserved—features of the DDR are the and Weinert 1995; Rouse and Jackson 2002a). Conse- recruitment of DNA-repair proteins to sites of DNA quently, it is probably best to consider the DDR as an damage and the “checkpoint” events that slow down or integrated and highly coordinated set of events. These arrest cell-cycle progression, thus delaying key cell-cycle issues should therefore be borne in mind in the sections transitions until the damage has been removed (Zhou below where, for the sake of simplicity, we summarize and Elledge 2000; Khanna and Jackson 2001). Once the the key features of DNA repair and DNA-damage check- DNA damage has been repaired, the blocks to cell-cycle point events separately and then discuss how each set of progression are relieved and cell proliferation can re- factors impacts on telomere biology.

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Telomeres and the DNA-damage response

DNA-damage checkpoint pathways evant PIKK and for this to phosphorylate a subset of its targets. One such target is the C terminus of the histone To the first approximation, DNA-damage checkpoint H2A variant H2AX (H2A in S. cerevisiae), which is phos- events can be likened to a classical intracellular signal- phorylated extensively in the chromatin flanking sites of transduction pathway. Thus, the “stimulus” (DNA dam- DNA damage (Nussenzweig 2004). The resulting phos- age) is detected by a “sensor” (DNA-damage-binding pro- phorylated species of H2AX, referred to as ␥-H2AX, is tein) that then triggers the activation of a “transduction” then thought to facilitate the DDR by inducing changes system composed of upstream (proximal) and down- in local chromatin structure and by facilitating the focal stream (distal) protein kinases, together with a series of accumulation of DNA-repair and checkpoint proteins to adaptor proteins (Fig. 2). This kinase cascade amplifies the damaged regions. the initial DNA-damage signal and triggers a diverse set 2Notably, however, the phosphorylation of other of outputs through targeting a range of “effector” pro- checkpoint PIKK targets also requires additional factors, teins. Central to the DDR in all organisms studied are at least some of which may be classified as DNA-damage two large and highly conserved protein kinases of the sensors as they are recruited to sites of DNA damage PIKK (phosphatidyl inositol 3-kinase-like kinase) family. independently of the PIKK-containing complexes. In hu- In humans these “checkpoint PIKK” proteins are termed mans, these additional factors include the replication ATM and ATR (ATM and RAD3-related), whereas in S. factor C (RF-C)-like complex containing hRAD17 in as- cerevisiae and S. pombe they are known as Tel1p and sociation with the small RF-C subunits, and the prolif- Mec1p, respectively, and Tel1p and Rad3p, respectively erating cell nuclear antigen (PCNA)-like hRAD9– (see Table 2). The available evidence indicates that the hRAD1–hHUS1 (9–1–1) complex (Shiomi et al. 2002; for two kinases have distinct but partially overlapping func- review, see Karnitz 2004; see Table 2 for yeast orthologs). tions. Thus, mammalian ATM is involved primarily in Although other models for their actions exist, the sensing and responding to DNA double-strand breaks PCNA- and RF-C-like checkpoint complexes might pro- (DSBs) generated by agents such as ionizing radiation, mote the DDR by enhancing the activity of the check- although in the absence of ATM some of these functions point PIKK proteins and/or by recruiting checkpoint are partly assumed by ATR (Shiloh 2003). By contrast, PIKK substrates to the vicinity of DNA damage, thus ATR responds to a wider range of lesions, probably after facilitating their phosphorylation. Finally, efficient they have been processed to a common single-stranded checkpoint activation also requires the recently charac- DNA intermediate, and is particularly important in re- terized “mediator” proteins, which include mammalian sponding to DNA damage during S phase (Zou and BRCA1, 53BP1, MDC1/NFBD1, and Claspin, together Elledge 2003). Once activated, the checkpoint PIKK pro- with yeast counterparts such as S. cerevisiae Rad9p (Shi- teins phosphorylate a range of factors including the dis- loh 2003; for review, see Stucki and Jackson 2004). One tal checkpoint kinases CHK1 and CHK2 (Chk1p and function of these proteins appears to be to facilitate the Rad53p in S. cerevisiae; Chk1p and Cds1p in S. pombe) focal accumulation of checkpoint and DNA-repair fac- that then target various effector proteins involved in tors in damaged regions, thus promoting their phos- modulating DNA repair, transcription, and cell-cycle phorylation and leading to more efficient checkpoint ac- progression (Bartek and Lukas 2003). tivation and DNA repair (e.g., Gilbert et al. 2001; Gold- Precisely how the checkpoint-PIKKs are activated by berg et al. 2003). DNA damage is still open to debate. One study suggested that several stresses that induce chromatin alterations in DNA-repair pathways the absence of DSBs can lead to ATM autophosphoryla- tion and activation (Bakkenist and Kastan 2003). Other Different DNA-damaging agents tend to yield chemi- reports have suggested that DSBs trigger efficient ATM cally distinct classes of lesions and, generally speaking, activation after they have been bound and/or nucleolyti- each class of lesions is repaired by one or more distinct cally processed by the MRE11–RAD50–NBS1 (MRN) DNA-repair pathways (Friedberg et al. 1995). Of particu- complex (D’Amours and Jackson 2002; Uziel et al. 2003; lar importance in regard to telomere functions are the Weizman et al. 2003). Consistent with such a model, two principal pathways of DNA DSB repair: homologous work in S. cerevisiae has shown that the analogous recombination (HR) and nonhomologous end-joining Mre11p–Rad50p–Xrs2p (MRX) complex promotes Tel1 (NHEJ). Both of these systems have been highly con- activation (D’Amours and Jackson 2001; Usui et al. served throughout eukaryotic evolution but, whereas 2001). On the other hand, ATR activation requires its NHEJ is a major pathway for DNA DSB repair in higher associated regulatory subunit ATR-interacting protein eukaryotes, single-celled organisms such as yeast rely (ATRIP; Lcd1p/Ddc2p in S. cerevisiae and Rad26p in S. most heavily on HR (Lieber et al. 2003; Sung et al. 2003). pombe; see Table 2). One substrate for such complexes is HR requires the RAD52 epistasis group of genes and in- replication protein A (RPA)-coated single-stranded DNA volves the damaged DNA entering into synapsis with an (Zou and Elledge 2003), although evidence has also been undamaged homologous partner. An early event in HR is provided for direct DNA binding by these complexes the resection of the DNA DSB in the 5Ј-to-3Ј direction by (Rouse and Jackson 2002b; Bomgarden et al. 2004; Unsal- a nuclease, whose activity appears to be modulated by Kacmaz and Sancar 2004). On their own, the above com- the MRN complex. The resulting 3Ј single-stranded plexes appear to be sufficient for activation of the rel- DNA tails are then bound by Rad51p (a process that is

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Table 2. Proteins involved in checkpoint–PIKK pathways are conserved from yeast to man

Checkpoint factors characteristics S. cerevisiae S. pombe Human

Upstream kinases (PIKKs) Mec1p Rad3p ATR Tel1p Tel1p ATM PIKK-interacting subunit Lcd1p/Ddc2p Rad26p ATRIP Signal-modifier Mre11p–Rad50p–Xrs2p Rad32p–Rad50p–Nbs1p MRE11–RAD50–NBS1 RFC-like subunit (clamp-loader) Rad24p Rad17p RAD17 PCNA-like subunit (sliding clamp) Rad17p Rad1p RAD1 ‘ Ddc1p Rad9p RAD9 Mec3p Hus1p HUS1 Mediators Rad9p Crb2p 53BP1, MDC1? Dpb11p Cut5p TOPBP1? BRCA1 Mrc1p Mrc1p Claspin Downstream transducer kinases Chk1p Chk1p Chk1 Rad53p and Dun1p Cds1p CHK2/CDS1

facilitated by a range of other HR factors), which cata- sion and ensuing loss of proliferative capacity (Naito et lyzes a strand-exchange reaction with a homologous un- al. 1998; Nakamura et al. 1998, 2002; Ritchie et al. 1999). damaged DNA molecule. Subsequently, the 3Ј terminus An analogous analysis of mammalian cells defective in of the damaged molecule is extended by DNA polymer- both ATM and ATR has not been possible because ATR ase, ligation takes place and the DNA crossovers (Holli- is essential for cell viability (Brown and Baltimore 2000). day junctions) are resolved to yield two intact DNA mol- Notably, it has recently been observed that Tel1p and ecules. By contrast, NHEJ does not require an undam- Mec1p are alternatively associated with the telomere aged partner molecule and essentially any two exposed during the S. cerevisiae cell cycle—Mec1p peaking in S double-stranded DNA ends can be re-ligated. In all eu- phase and Tel1p in the other phases—and that Mec1p karyotic species examined, NHEJ involves the heterodi- kinase activity governs this association (Takata et al. meric DNA end-binding protein Ku together with DNA 2004). Furthermore, in the absence of Tel1p, Mec1p as- ligase IV in association with a regulatory subunit sociates with the telomere throughout the cell cycle. (XRCC4 in mammals). In vertebrates, efficient NHEJ Taken together, these observations reveal a crucial role also requires the DNA-dependent protein kinase cata- for the yeast checkpoint PIKKs in telomere mainte- lytic subunit (DNA-PKcs; a member of the PIKK family), nance, and suggest that these two kinases act in two which is targeted to DNA DSBs by Ku (Smith and Jack- distinct telomere maintenance pathways that can par- son 1999; Downs and Jackson 2004). In many cases, tially compensate for one another. NHEJ also involves additional proteins that help the pro- Mounting evidence suggests that the checkpoint cessing of DNA ends prior to their ligation (Lieber et al. PIKKs act in analogous ways at the telomere and in the 2003). As discussed further below, proteins associated DDR. For example, in all cases examined both roles re- with certain other DNA-repair pathways have also been quire the integrity of the PIKK kinase catalytic domain implicated in telomeric functions. (Greenwell et al. 1995; Mallory and Petes 2000). Further- more, in line with functional interactions between S. cerevisiae Tel1p and the MRX complex in the DDR DNA-damage checkpoint proteins and telomeres (D’Amours and Jackson 2001; Usui et al. 2001), Tel1p One of the most compelling indications for a central role and MRX also work in the same pathway of telomere of DNA-damage checkpoint factors at telomeres is the length maintenance (Boulton and Jackson 1998; Nugent observation that inactivation of checkpoint PIKKs leads et al. 1998; Ritchie and Petes 2000; Gallego and White to major defects in telomere length control and telo- 2001; Ranganathan et al. 2001). That is, the loss of any meric stability in all organisms examined. For example, one of these proteins causes telomere shortening to a inactivation of Tel1p in S. cerevisiae, Rad3p in S. pombe new, stable, length, but no further shortening is observed or ATM in human cells causes telomere shortening with compound mutants, at least as detectable with the (Lustig and Petes 1986; Greenwell et al. 1995; Metcalfe available techniques. Moreover, as with inactivation of et al. 1996; Dahlen et al. 1998; Naito et al. 1998; Mat- TEL1, disruption of RAD50 in a mec1 mutant back- suura et al. 1999; Hande et al. 2001). In the above yeast ground leads to dramatic telomere shortening and ensu- mutants, the telomeres initially shorten rapidly but then ing growth arrest (Ritchie and Petes 2000). In S. pombe, stabilize at a new, shorter length. By contrast, the com- inactivation of either RAD3 or RAD26—which encodes pound inactivation of both checkpoint PIKKs in S. cer- the regulatory subunit of Rad3p in the DDR—causes evisiae or S. pombe leads to a total inability to maintain similar telomere shortening (Naito et al. 1998; Naka- telomeric tracts by telomerase-dependent mechanisms, mura et al. 2002). Furthermore, despite the loss of S. thus causing dramatic and progressive chromosome ero- pombe Tel1p or orthologs of the MRX complex having

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Telomeres and the DNA-damage response modest, if any, effect on telomere length (Wilson et al. type that is similar to that of mec1 tel1 and mec1 mre11 1999; Hartsuiker et al. 2001; Manolis et al. 2001; Naka- mutants (Mieczkowski et al. 2003). Similarly, compo- mura et al. 2002; Ueno et al. 2003), combining their loss nents of the analogous S. pombe PCNA- and RF-C-like with inactivation of RAD3 leads, in each case, to an in- complexes influence telomeres via the RAD3/RAD26 ability to maintain telomeres by telomerase-dependent pathway but their loss does not fully recapitulate the mechanisms (Ritchie et al. 1999; Nakamura et al. 2002; phenotypes of RAD3-orRAD26-deficient strains (Naka- Chahwan et al. 2003). This phenotype is also observed in mura et al. 2002). Some other less well-characterized S. pombe strains deleted for TEL1 and RAD26 (Naka- DDR factors also regulate telomere functions. For ex- mura et al. 2002). Finally in this regard, RPA—which ample, S. cerevisiae Tel2p works in the same telomere facilitates the recruitment of mammalian ATR–ATRIP maintenance pathway as Tel1p (Runge and Zakian 1996) and S. cerevisiae Mec1p–Ldc1p/Ddc2p to single-stranded and seems to bind to telomeric DNA (Kota and Runge DNA (Zou and Elledge 2003)—has been implicated in 1998). Although a role of Tel2p in the DDR has not yet telomere length control (Smith et al. 2000; Mallory et al. been described, its human counterpart controls sensitiv- 2003) and in controlling the access of Est1p to the telo- ity to DNA damaging agents whereas its C. elegans or- mere (Schramke et al. 2004). Taken together, the avail- tholog influences telomere length, the S-phase check- able data therefore strongly suggest that triggering point, and controls life span and biological rhythms checkpoint PIKK activity is necessary for normal telo- (Ahmed et al. 2001; Benard et al. 2001; Lim et al. 2001; mere homeostasis, and suggest that the mechanism by Jiang et al. 2003). which this occurs is closely related to the events leading It is interesting to note that the components of the to PIKK activation in the DDR. DDR that tend to have most impact at the telomere are Other upstream components of the DDR, particularly those that function in the upstream parts of the DDR potential sensors of DNA lesions, also impinge on telo- signalling cascade. Thus, while the checkpoint PIKKs mere length regulation. Perhaps the most compelling and factors involved in their regulation/activation have evidence for this is the observation that C. elegans major roles in telomere homeostasis, proteins that play strains lacking MRT2—a functional ortholog of human important but more downstream functions in the RAD1 that forms part of the 9–1–1 complex—display DDR—such S. cerevisiae Rad9p, Rad53p, Dun1p and progressive telomere shortening and loss of germ-line Chk1p—do not. Furthermore, in instances where such immortality (Ahmed and Hodgkin 2000). However, the downstream factors influence the telomere, this has gen- deletion of components of the analogous complex in S. erally been ascribed to an indirect effect. For example, cerevisiae causes only mild telomere length changes, the impact of RAD53 or DUN1 deletion on telomere and some effects appear to be laboratory or strain specific length seems to at least in part reflect defective regula- (Corda et al. 1999; Longhese et al. 2000; Grandin et al. tion of deoxyribonucleotide levels (Longhese et al. 2000; 2001a). There have also been contrasting reports on the Mallory et al. 2003). Where analyzed, downstream com- potential role of the analogous S. pombe complex in telo- ponents of the DDR in mammals, such as p53 and mere length regulation, although a recent extensive H2AX, have also not been found to have a major impact analysis concluded that these factors and Rad17p—a on telomere length regulation (Chin et al. 1999; Fernan- component of the RF-C-like checkpoint complex—do dez-Capetillo et al. 2003). Taken together, the available control telomere length and are associated with telo- data are therefore consistent with a model in which telo- meric DNA in vivo (Nakamura et al. 2002 and references mere homeostasis involves (certain) sensor and upstream therein). Although the mechanism(s) by which these fac- kinase components of the DDR that influence telomere tors influence telomere length regulation is still unclear, structure and telomerase action by mechanisms that do one possibility is that they facilitate the phosphorylation not require the actions of more downstream transducers of certain checkpoint PIKK targets involved in telomere or effectors of the DDR. maintenance. Alternatively, or in addition, the effects of Based on the above, it seems probable that checkpoint these factors on telomere length might reflect them al- PIKKs and their regulatory factors respond to a specific tering telomeric chromatin structure (Corda et al. 1999) DNA structure(s) arising at telomeres. One situation or the maturation of telomeric lagging-strand DNA rep- where such structures may occur is during S phase, when lication intermediates. It is noteworthy that S. cerevisiae telomeres are replicated and their specialized functions cells lacking an alternative RF-C-like checkpoint com- might be temporarily disrupted. In this regard, it is note- plex containing Elg1p have long telomeres (Kanellis et al. worthy that Tel1p and the MRX complex function to- 2003; Smolikov et al. 2004). gether in responding to DSBs during S phase (D’Amours Significantly, combining the deletion of TEL1 with de- and Jackson 2001; Grenon et al. 2001; Usui et al. 2001) letion of components of the PCNA-like checkpoint com- and that replication of telomeres may transiently pro- plexes in S. cerevisiae, and the PCNA- and RF-C-like duce similar structures. At the telomere, leading- and checkpoint complexes in S. pombe, do not result in the lagging-strand DNA replication are expected to produce senescent phenotypes observed with deletion of MEC1 a blunt end and a recessed end with a 3Ј overhang, re- and TEL1 or RAD3 and TEL1, respectively (Nakamura et spectively (Chakhparonian and Wellinger 2003). Al- al. 2002; Mieczkowski et al. 2003). However, telomere- though replication products bearing a 3Ј overhang might to-telomere fusions do occur with increased frequency in directly serve as a template for telomerase with little or S. cerevisiae ddc1 tel1 and mec3 tel1 mutants, a pheno- no processing needed, blunt-ended products would pre-

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d’Adda di Fagagna et al. sumably require extensive processing to generate the 3Ј Abl (a protein implicated in DNA-PK- and ATM-depen- overhang needed for the binding of telomeric single- dent DDR events) has been found to inactivate telomer- stranded DNA-binding proteins such as Cdc13p. In line ase activity (Kharbanda et al. 2000). Nevertheless, it with this, differential processing of the two products has seems unlikely that the checkpoint PIKK proteins con- been revealed by studies with S. cerevisiae strains lack- trol telomere length primarily by influencing intrinsic ing the Rad27p nuclease, which functions in DNA-base telomerase catalytic activity, since in vitro telomerase excision repair and in the processing of Okazaki-frag- activity is largely unaffected by their loss and in S. cer- ment DNA-replication intermediates (Parenteau and evisiae the targeting of telomerase to telomeres by way Wellinger 2002). of a protein fusion rescues the senescent phenotype of The use of a de novo telomere addition assay employ- mec1 tel1 mutant cells (Chan et al. 2001). Therefore, it ing a telomeric DNA substrate bearing a HO-endonucle- seems most likely that checkpoint PIKK proteins such as ase-induced 5Ј overhang has revealed an involvement of Tel1p and Mec1p mainly control telomere length by MRX for telomerase action and Cdc13p binding to the de regulating the access of telomerase to telomeres by tar- novo substrate (Diede and Gottschling 2001). Based on geting additional telomere-bound factors. these results, it was proposed that the MRX complex Some potential telomeric targets for the checkpoint helps to prepare telomeric DNA for the loading of PIKKs in yeast have arisen through the work of D. Shore Cdc13p, which then protects the chromosome from fur- and collaborators, who demonstrated that telomere elon- ther degradation and recruits telomerase and other DNA gation by telomerase is progressively inhibited in cis by replication components to synthesize telomeric DNA. telomere-bound Rap1p. In this elegant model of telomere However, in apparent opposition to this model, the as- length homeostasis (the so-called Rap1 counting mecha- sociation of Cdc13p with natural yeast telomeres was nism; Marcand et al. 1997), progressive telomere short- found to occur efficiently in the absence of Tel1p or ening causes the gradual loss of telomere-bound Rap1p MRX and moreover, mutations in the exonuclease do- and, therefore, a progressive relief of its inhibitory func- main of Mre11p did not affect telomere length (Moreau tion on telomerase activity, ultimately resulting in et al. 1999; Tsukamoto et al. 2001). The recent finding telomerase-mediated telomere elongation. Significantly, that the MRX complex does play a modest but detectable this Rap1 counting mechanism does not function in the role in the generation of telomere overhangs outside S absence of Tel1p and, furthermore, the deletion of the phase could reconcile the above observations (Larrivée et Rap1p-binding factors, Rif1p and Rif2p, leads to telom- al. 2004). In addition, it is also possible that the MRX erase-dependent telomere elongation in wild-type but complex acts in a partially redundant manner with other not in tel1 mutant cells (Craven and Petes 1999; Ray and proteins at the telomere; one such protein might be the Runge 1999). Taken together, these results suggest a conserved exonuclease Exo1p, which in S. cerevisiae model in which Tel1p and the Rap1p/Rif1p/Rif2p com- regulates single-stranded telomeric DNA degradation in plex promote telomere elongation by acting in the same the absence of Ku (Maringele and Lydall 2002). Further genetic pathway. Notably, the human homolog of evidence that the yeast MRX complex is involved in re- Rap1p, hRAP1, does not appear to bind DNA directly but cruiting telomerase activity to telomeres is provided by instead acts together with the telomere-specific DNA- the observation that robust telomere lengthening takes binding protein TRF2 to negatively regulate telomere place in mec1 mrx and mec1 tel1 mutant cells in situa- length in a telomerase-dependent fashion (Li and de tions where telomerase is targeted to telomeres by way Lange 2003). The recent discovery of mammalian ortho- of a protein fusion (Tsukamoto et al. 2001). Such a role logues of Rif1p (Adams and McLaren 2004) and the sur- may also exist in mammals, as NBS1 associates with prising finding that human Rif1 plays important roles in telomeres during S phase when telomeres are elongated the DDR but seemingly not in telomere homeostasis (Zhu et al. 2000), and is required for effective telomere (Silverman et al. 2004) adds further potential layers of elongation by telomerase (Ranganathan et al. 2001). complexity to their functions. Although there are many ways in which the check- point PIKKs and associated components may influence DNA-repair proteins and telomeres telomere homeostasis, these can be reconciled with a model in which such factors regulate telomerase activity One of the first indications for an involvement of DNA- or telomerase access to the telomeric template. One pos- repair factors in normal telomeric functions was the dis- sibility, discussed above, is that such factors are needed covery in 1996 that inactivation of either subunit of the for the efficient processing of nascent telomeres into NHEJ protein Ku leads to telomere shortening in S. cer- structures compatible with telomerase action. In addi- evisiae (Boulton and Jackson 1996; Porter et al. 1996). It tion, several lines of evidence suggest that they might was subsequently shown that inactivation of Ku also also influence telomerase activity more directly. For ex- triggers the rapid loss of telomeric repeats from chromo- ample, ionizing radiation can influence hTERT nuclear some termini in S. pombe (Baumann and Cech 2000). localization (Wong et al. 2002), and telomerase activity However, contrary to when telomerase components are was found to increase in extracts derived from rodent deleted, these telomeres stabilize at a new, shorter, cells that had been treated with ionizing radiation or length and there is no progressive further telomere attri- ultra-violet light (Hande et al. 1997, 1998). Conversely, tion leading to loss of cell proliferation (Boulton and DNA-damage-induced phosphorylation of hTERT by c- Jackson 1998; Nugent et al. 1998; Polotnianka et al.

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Telomeres and the DNA-damage response

1998; Baumann and Cech 2000). Notably, whereas telo- have revealed that Ku physically associates with mam- mere shortening is also caused by the loss of S. cerevisiae malian telomeres in vivo (Hsu et al. 1999; d’Adda di Mre11p, Rad50p, or Xrs2p (which also function in Fagagna et al. 2001). In addition, inactivation of one al- NHEJ), this is not the case when S. cerevisiae DNA- lele of the gene for Ku80 in human cells results in telo- ligase 4 or Lif1p (the XRCC4 homolog) are inactivated mere shortening (Myung et al. 2004). Furthermore, inac- (e.g., Teo and Jackson 1997; Boulton and Jackson 1998; tivation of both alleles leads to cell death, although it is Herrmann et al. 1998; D’Amours and Jackson 2002). not clear whether this is due to further telomere short- Consistent with these findings, the role of Ku at telo- ening or an inability to cope with endogenous DNA meres appears to be distinct from its roles in NHEJ, as Ku damage (Li et al. 2002). Differently, Ku inactivation is mutants have been identified that affect one function not lethal in mice. Although the reason for this differ- but not the other (Driller et al. 2000; Bertuch and Lund- ence between humans and mice is not clear, it has been blad 2003; Stellwagen et al. 2003; Roy et al. 2004). observed that the Ku80 locus expresses a primate-spe- Chromatin immunoprecipitation and immunolocal- cific alternative form of the protein, known as KARP-1, ization studies have shown that Ku is physically associ- that is absent in rodents (Myung et al. 1997). In mice, the ated with telomeres in both S. cerevisiae and S. pombe analysis of the role of Ku at telomeres has generated (Gravel et al. 1998; Laroche et al. 1998; Nakamura et al. some contrasting conclusions. One study showed that 2002), although it is not yet clear whether this reflects cells derived from transgenic mice lacking Ku80, and direct binding of Ku to telomeric DNA or it being teth- embryonic stem cells lacking Ku70, have shorter telo- ered by protein–protein interactions, or both. One meres than their controls, while cells lacking Ligase IV mechanism by which Ku functions at the telomere has or XRCC4 do not display marked telomere length alter- been revealed by work showing that S. cerevisiae Ku ations (d’Adda di Fagagna et al. 2001). This report also regulates telomere length by interacting directly with showed that Ku inactivation causes elevated chromo- TLC1 (Peterson et al. 2001; Stellwagen et al. 2003). In- somal instability, leading to chromosomal fusions that deed, overexpression of a conserved stem loop of TLC1 generally lacked detectable telomeric repeats at the fu- leads to Ku-dependent telomere shortening, deletion of sion sites. By contrast, a report from another group ob- this stem loop causes telomere shortening, and a YKU80 served that inactivation of Ku80 did not lead to telomere mutation that renders Ku unable to bind TLC1 results in shortening and that the chromosomal fusions retained short telomeres (Stellwagen et al. 2003). Taken together, telomeric DNA at the fusion points (Samper et al. 2000). these data suggest that the binding of Ku to the telom- Furthermore, an additional report from the same group erase RNA and perhaps other telomere-specific proteins suggested that Ku is a negative regulator of telomere ac- plays a key role in ensuring that telomerase is targeted cess by telomerase (Espejel et al. 2002a). Since both appropriately to chromosomal ends (Fig. 1). Signifi- groups analyzed mice with the same genetic deletion, cantly, the deletion of Ku also impairs the synthesis and/ the differences reported may originate from variations in or stability of chromosomal termini in S. cerevisiae. the experimental procedures of telomere length mea- Thus, whereas the telomeric 3Ј overhang is detectable surement, or from differences in mouse or cell mainte- only during S phase in wild-type cells, in Ku mutants nance. Importantly, both analyses found that Ku inacti- these overhangs are observed in all cell-cycle phases vation does not lead to the dramatic changes in telo- (Gravel et al. 1998). This has lead to the suggestion that meric overhangs that are observed in yeast. the lack of Ku leads to a defect in lagging-strand DNA Perhaps surprisingly, inactivation of Ku in Arabidop- replication of the telomere (Gravel and Wellinger 2002) sis thaliana was found to lead to telomerase-dependent and a lack of protection towards Exo1p and other exo- telomere lengthening and inefficient C-strand mainte- nucleases, resulting in the generation of the observed nance (Bundock et al. 2002). However, the compound constitutive overhang (Maringele and Lydall 2002). In S. inactivation of Ku and telomerase in A. thaliana causes cerevisiae, Ku is also required for transcriptional silenc- a faster rate of telomere shortening than telomerase in- ing at telomeres (Tsukamoto et al. 1997; Boulton and activation alone (Riha et al. 2002; Riha and Shippen Jackson 1998)—a function that may in part reflect inter- 2003). Significantly, the deletion of MRE11 also caused actions between Ku and SIR proteins (Tsukamoto et al. telomere elongation in A. thaliana (Bundock and 1997; Roy et al. 2004)—and for tethering telomeres to the Hooykaas 2002). Although these findings were unex- nuclear periphery (Laroche et al. 1998). Such tethering pected, it is noteworthy that while telomerase inactiva- may limit HR between telomeres (Polotnianka et al. tion restricts life span in most organisms, it extends life 1998) and ensure that telomeres are replicated in late S span in A. thaliana (Riha et al. 2001). A unifying model phase (Cosgrove et al. 2002). for the telomeric functions of Ku in different species is Several lines of evidence indicate that Ku also func- further complicated by the observation that inactivation tions in telomere maintenance in mammals. For ex- of Ku in chicken DT40 cells does not seem to affect ample, it has been reported that human Ku interacts telomere length (Wei et al. 2002). with both TRF1 and TRF2 (Hsu et al. 2000; Song et al. In the mouse, inactivation of the Ku-associated NHEJ 2000; Peterson et al. 2001), suggesting that it may coop- protein, DNA-PKcs, leads to telomere fusions in the ab- erate with these proteins to regulate telomere length and sence of detectable telomere shortening, suggesting that establish telomere end-protection, respectively. In line it may be involved in telomere capping (Bailey et al. with this idea, chromatin immunoprecipitation studies 2001; Gilley et al. 2001; Espejel et al. 2002b). Consistent

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d’Adda di Fagagna et al. with this idea, DNA-PKcs is associated with telomeric cation machinery to fully replicate chromosomal ter- DNA in human cells (d’Adda di Fagagna et al. 2001) and mini. Consequently, telomeres progressively shorten inhibition of DNA-PKcs catalytic activity by chemical upon repeated cell divisions, ultimately becoming so inhibitors results in telomere fusions in human cells short that their normal functions are perturbed. It is still (Bailey et al. 2004). Finally, it has been shown that mice unclear what is the minimal length below which a telo- lacking DNA-PKcs and Terc display faster rates of telo- mere triggers a DDR. Recently, it has been shown that a mere loss than mice lacking Terc alone (Espejel et al. DDR at critically short telomeres is associated with the 2002b). absence of TRF2, at least as detected by immunofluores- Whether proteins associated with HR also have key ence experiments (Herbig et al. 2004), suggesting that the functions at normal telomeres is still unclear. Thus, recruitment of this protein to a telomere could be the while loss of RAD52 or RAD51 does not affect telomere limiting factor. In some cell types telomere dysfunction length in S. cerevisiae, rad52 tlc1, rad51 tlc1, or rad52 lead to apoptosis whereas in others, such as human fi- est1 double mutant cells senesce at a faster rate than tlc1 broblasts, it triggers a permanent growth arrest called or est1 single mutants (Lundblad and Blackburn 1993; Le senescence. Recent work has established that telomere- et al. 1999) and Rad54 knock-out mice have recently initiated senescence shares many features of a cell-cycle been shown to bear shorter telomeres than matched con- arrest induced by DNA-damaging agents that cause trols (Jaco et al. 2003). Furthermore, Rad51 inactivation DSBs (d’Adda di Fagagna et al. 2003). These include the in chicken DT40 cells has been reported to increase the activation of upstream checkpoint PIKKs, mediators, presence of the telomeric overhangs (Wei et al. 2002). and downstream kinases of the DDR, and the appearance Most recently, it was established that the RAD51-related of senescence-associated DNA damage foci (SDFs) con- protein RAD51D colocalizes with telomeres in human taining DDR factors, as detected by immunofluores- cells and that inactivation of this factor leads to cell cence; one report, however, concluded that the detect- death, possibly as a consequence of telomere uncapping ability of such markers is only transient (Bakkenist (Tarsounas et al. 2004). In light of these findings, it is et al. 2004). The appearance of DDR markers in senes- tempting to speculate that RAD51D, possibly in a com- cent cells is triggered with the direct contribution of plex with certain other HR factors, promotes telomere eroded telomeres, as revealed by the specific accumula- T-loop formation. In addition, and as discussed below, tion of ␥-H2AX and other markers of the DDR at chro- HR factors can play key roles in maintaining telomere mosome termini in senescent cells. Significantly, inter- length by telomerase-independent mechanisms. fering with the actions of checkpoint kinases by siRNA Other DNA-repair proteins have also been implicated or by dominant-negative constructs leads to a significant in telomere maintenance. For example, the mammalian proportion of senescent cells resuming cell cycle progres- DNA repair protein poly(ADP-ribose) polymerase sion into S phase, indicating that DNA-damage check- (PARP-1)—which functions in DNA base-excision repair point activation is causally associated with the senes- and single-strand break repair (D’Amours et al. 1999)— cent state (d’Adda di Fagagna et al. 2003; Herbig et al. acts at the telomere. Indeed, a study of PARP-1 knock- 2004). out mice provided the first demonstration of a protein of Similarly, progressive telomere shortening caused by the DDR functioning at the telomere in vertebrates inactivation of telomerase in S. cerevisiae leads to the (d’Adda di Fagagna et al. 1999). In this report, PARP-1 accumulation of cells that are unable to divide further inactivation was shown to lead to stable, shortened, telo- and which display an activated DDR—as determined by meres and genomic instability in two different mouse the phosphorylation of Rad53p—and a morphology remi- genetic backgrounds and in different tissues. Further- niscent of senescent mammalian cells (Enomoto et al. more, the compound inactivation of PARP-1 and p53 2002; IJpma and Greider 2003). Moreover, inactivation of lead to very long and heterogeneous telomeres (Tong et checkpoint factors such as Mec3p, Mec1p, Lcd1p/Ddc2p, al. 2001), perhaps reflecting the ability of both PARP-1 or Rad24p allows a portion of such cells to bypass this and p53 to suppress HR (Mekeel et al. 1997; Schultz et al. senescence-like state and continue proliferating. There- 2003). However, a different group reported that PARP-1 fore, as in mammalian cells, severe telomere shortening inactivation does not affect telomere length (Samper et in yeast leads to the activation of the DDR and concomi- al. 2001). The use of two different genetic deletions in tant cell-cycle arrest. These findings are consistent with two different mouse strains may help to explain these biochemical experiments and micro-array expression apparently contradictory results. Finally, XPF/XRCC1— analyses, which have shown that yeast cells with criti- which interacts with ERCC1 to form a structure-specific cally short telomeres have a global gene expression pro- endonuclease involved in nucleotide excision repair file that overlaps with that of cells exposed to DNA- (de Laat et al. 1999)—was recently shown to regulate damaging agents (Nautiyal et al. 2002). Furthermore, the the stability of the telomeric 3Ј overhang (Zhu et al. observation that mice with shortened telomeres are 2003). more sensitive to radiation (Goytisolo et al. 2000; Wong et al. 2000) is consistent with a model in which dysfunc- tional telomeres are perceived as DSBs and therefore DDR proteins at dysfunctional telomeres cells bearing them are more sensitive to additional DNA Most human somatic cells do not express sufficient damaging agents generating DSBs. Taken together, these telomerase to cope with the inability of the DNA repli- results suggest that eroded telomeres and DNA damage

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Telomeres and the DNA-damage response trigger very similar responses and ultimately produce (Baumann and Cech 2001; Ferreira and Cooper 2001; similar outcomes. Smogorzewska et al. 2002; Mieczkowski et al. 2003). No- Telomere shortening is not the only way the protec- tably, however, there are suggestions that differences ex- tive function of the telomere can be lost. In mammals, ist between the mechanism of telomere end fusions and removal of TRF2 from the telomere leads to a DDR that NHEJ of DNA DSBs caused by DNA-damaging agents. results in cell-cycle arrest or apoptosis, depending on the For example, although S. cerevisiae Nej1p—an essential cell type (van Steensel et al. 1998). Moreover, the DDRs NHEJ component—does not affect the stability of telo- in senescent and TRF2-inhibited cells appear to be strik- meres in wild-type cells, it suppresses telomere fusions ingly similar (d’Adda di Fagagna et al. 2003; Takai et al. mediated by NHEJ in yeasts maintaining their telomeres 2003). Taken together, these results suggest that the loss via HR (Liti and Louis 2003). of telomeric DNA is not detrimental per se, but it is the When telomeres become critically short in the absence loss of telomere-bound factors that results in telomere of telomerase in S. cerevisiae, rare survivors emerge that deprotection and concomitant activation of the DDR. maintain their telomeres through RAD52-dependent This idea is further supported by the observation that mechanisms of HR (Lundblad and Blackburn 1993; Le et cells senesce with a shorter mean telomere length if al. 1999). These survivors employ either RAD50-depen- TRF2 is overexpressed; presumably the additional TRF2 dent amplification of TG-repeats (type II recombination) helps to stabilize short telomeres (Karlseder et al. 2002). or RAD51-dependent acquisition of subtelomeric ele- Analogously, inactivation of S. cerevisiae CDC13, STN1, ments (and their deletion derivatives) by a large number or TEN1—which form a complex that binds to and pro- of telomeres (type I recombination; Lundblad and Black- tects the protruding telomeric 3Ј overhang—leads to dra- burn 1993; Teng and Zakian 1999; Teng et al. 2000; Chen matic activation of the DDR (Garvik et al. 1995; Grandin et al. 2001; for review, see Lundblad 2002). It is notewor- et al. 1997, 2001b; Pennock et al. 2001). In addition, a thy that, although such events might occur most com- DDR leading to rapid telomere degradation has been ob- monly on telomeres that either have lost telomerase ac- served in S. pombe lacking Pot1p—a telomeric single- tivity or Ku (McEachern et al. 2000), recombination can stranded protein similar to those found in ciliated pro- also occur on long telomeres that have been uncapped by tozoa (Baumann and Cech 2001). Whether human Pot1p the loss of Cdc13p, suggesting that these factors protect has a similar protective role, however, is still unclear chromosome ends from such reactions (Booth et al. 2001; (Colgin et al. 2003; Loayza and de Lange 2003). Grandin et al. 2001a; DuBois et al. 2002; Tsai et al. 2002; Perhaps unexpectedly, unregulated telomere lengthen- Grandin and Charbonneau 2003). As mentioned previ- ing can also induce a DDR, as has been observed in S. ously, the loss of telomerase function in S. pombe leads cerevisiae cells bearing short telomeres and overexpress- to chromosomal circularization in surviving cells (Bau- ing Tel1p (Viscardi et al. 2003), and also can cause ge- mann and Cech 2000). However, when Taz1p is also de- nome instability and telomere fusions, as observed in S. leted in such backgrounds, the ensuing survivors more pombe cells lacking Taz1p (Ferreira and Cooper 2001). frequently use recombinational modes for telomere Furthermore, in human cells the overexpression of a hu- maintenance (Nakamura et al. 1998). Thus, as in S. cer- man ortholog of yeast Est1p—a factor necessary for evisiae, telomere end-protection proteins actively in- telomerase mediated telomere elongation (Snow et al. hibit HR among homologous telomeric sequences in S. 2003)—leads to telomere uncapping (Reichenbach et al. pombe. 2003). Although the mechanisms that trigger the DDR In mammals, a significant but relatively small portion under these circumstances are still unclear, it is possible of tumours (mostly sarcomas), and cell lines transformed that the uncoupling of the synthesis of the two strands, by the SV40 virus, show a very heterogeneous telomeric caused by an overactive telomerase, might lead to gen- pattern with some very long telomeres (Neumann and eration of an excess of single-stranded DNA that triggers Reddel 2002). These cells do not express detectable a DDR. Overall, these observations reveal that a variety telomerase and are believed to maintain their telomeres of perturbations of telomere structure can trigger a DDR by HR, as demonstrated by their ability to amplify a very similar to that caused by exogenous DNA-damaging tagged subtelomeric sequence in trans onto other chro- agents. mosomal termini (Dunham et al. 2000; Niida et al. 2000; Dysfunctional telomeres are not only substrates for Varley et al. 2002). Significantly, a portion of cells main- the cell-cycle checkpoint machinery but are also tar- taining telomeres by this “ALT” mechanism (for alter- geted by the DNA-repair apparatus. Indeed, in both native lengthening of telomeres) display evidence of a mammals and yeast, critically short telomeres are sub- DDR at some telomeres. In these cells, telomere-specific strates for recombination and are prone to telomere–telo- binding proteins and telomeric DNA—possibly includ- mere fusions. This leads to frequent chromosomal circu- ing this in an extra-chromosomal form—colocalize in larization in S. pombe cells lacking telomerase (Naka- subnuclear structures known as PML bodies together mura et al. 1998), and chromosomal aberrations with proteins usually associated with DNA damage resulting from chromosome end fusions in human fibro- checkpoint signalling and HR such as MRE11, NBS1, blasts approaching replicative senescence and in late RAD50, RAD51, RAD52, RPA, BLM, and WRN (Henson generation telomerase-deficient mice (Blasco 2002). et al. 2002). Although care should be used to interpret Similarly, uncapped telomeres are substrates for end- these colocalization data, as very long telomeres might joining events that involve well-known NHEJ factors render DDR proteins that are normally associated with

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d’Adda di Fagagna et al. telomeres more detectable than when telomeres are nuclear regions these proteins might physically prevent shorter, it is tempting to speculate that such structures DDR factors from gaining access to telomeric DNA. Ex- represent sites where telomeres are being maintained by amples of such activities might include single-stranded HR-based mechanisms. How cells become able to main- telomeric DNA-binding proteins with telomere protect- tain their telomeres in this manner is still open to con- ing functions, factors promoting T-loop formation, the jecture. Cell-fusion experiments suggest that ALT cells generally compact and repressive state of telomeric chro- generally carry a recessive mutation(s) (Neumann and matin and the localization to the nuclear periphery of Reddel 2002). Furthermore, circumstantial evidence sug- telomeres in some species. gests that p53 suppresses ALT, as cell lines derived from Nevertheless, the observation that some DDR factors Li-Fraumeni syndrome patients—which bear inherited are associated with telomeres and are necessary for p53 mutations—are frequently ALT, as are SV40 trans- proper telomere homeostasis suggests a more complex formed cell lines in which p53 activity has been essen- regulatory mechanism than mere exclusion of access. tially ablated. The observation that p53 negatively regu- Studies in S. cerevisiae have indicated that the induction lates HR (Mekeel et al. 1997), possibly by inhibiting of a full DDR is triggered by an unrepaired and most RAD51 activity (Linke et al. 2003), lends further support likely resected DSB (Pellicioli et al. 2001; Rouse and to this idea. Finally, it is possible that changes in telo- Jackson 2002a). Consistent with this idea, ATM activa- meric chromatin are associated with the assumption of tion in mammalian cells is compromised in cells im- ALT. For example, a change in telomeric chromatin that paired in the MRN nuclease complex (Uziel et al. 2003). made it more open and accessible to HR proteins could Moreover, at least for mammalian ATR and S. cerevisiae render the cell more susceptible to the initiation of ALT. Mec1, it appears that single-stranded DNA must be In this regard, it is noteworthy that inactivation of the S. bound by RPA in order for efficient checkpoint activa- cerevisiae HHO1 gene, which encodes the linker histone tion to ensue (Zou and Elledge 2003). Therefore, a telo- Hho1p, makes it more easy for the yeast cell to enter into mere might only activate the DDR if it becomes signifi- HR-dependent mechanisms of telomere maintenance cantly resected (Maringele and Lydall 2002) and com- (Downs et al. 2003). Perhaps inactivation or deregulation plexed with a sufficient number of RPA molecules. of linker histones, or possibly other chromatin changes, Indeed, short tracts of single-stranded telomeric DNA do could lead to a similar situation in the mammalian sys- not appear to normally activate the DDR, as such struc- tem. tures are present in cycling cells and in S. cerevisiae lacking Ku, where a large increase in these structures is generated in the absence of a detectable DDR (Gravel et DDR proteins at functional and dysfunctional al. 1998). In these situations the binding of Cdc13p (or telomeres: what’s the difference? Pot1p in S. pombe; Mitton-Fry et al. 2002) to single- As discussed above, components of the DDR are neces- stranded telomeric tracts presumably prohibits these be- sary both for normal telomere homeostasis and for re- ing recognized by large amounts of RPA. Notably, work sponding to dysfunctional telomeres. For example, the in yeast has shown that a DSB generated near a telomeric checkpoint PIKKs are necessary both for telomere ho- tract is not resected as efficiently as one located else- meostasis and to mount a DDR in reaction to the dis- where in the genome, and that this difference depends on ruption of the telomere protective structure following Cdc13p (Diede and Gottschling 1999). Thus, telomere telomere shortening and/or telomere deprotection. Simi- bound factors such as Cdc13p can curtail the DDR by larly, Ku can protect normal telomeric ends from resec- both binding to single-stranded telomeric tracts and tion and ensuing end fusions, and yet the NHEJ appara- therefore competing with RPA, and also by restricting tus actually mediates chromosomal end-to-end fusions further DNA resection into adjacent nontelomeric se- caused by telomere dysfunction. A key challenge for the quences (Fig. 3). In addition to potentially explaining telomere field is to explain how the DDR apparatus dis- why telomeres are not normally recognized as DNA tinguishes between functional and dysfunctional telo- damage, such a model could also help to explain why meres and produces two very different outcomes. DNA damage generated at the telomere is generally less The most obvious difference between these two situ- easily repaired than that at other chromosomal sites (von ations is the amount and constitution of telomere-asso- Zglinicki 2002). ciated proteins. It is therefore possible that the protein One situation when a normal telomere might be par- complexes associated in a sequence-specific manner ticularly vulnerable to triggering the DDR is when it is with telomeric DNA have the ability to limit the DDR. replicated—a replication fork reaching the end of a chro- Thus, when too few (or none) of such proteins are at a mosome end will face a situation very similar to that telomere, the DDR would become unrestrained, leading encountered by a fork replicating a chromosome carrying to chromosomal end fusions, cell-cycle arrest and/or a DNA strand break. In addition to the above described apoptosis (Fig. 3). We envision several, not necessarily mechanisms, it is possible that telomere-bound factors mutually-exclusive mechanisms by which telomeric directly modulate the kinase activity of the upstream proteins might inhibit a full DDR being elicited from a kinases of the DDR. Although this may be so, check- functional telomere. By direct steric hindrance and/or by point PIKK activity is clearly needed for normal telomere facilitating the formation of higher-order telomeric DNA homeostasis. One attractive possibility then is that the structures, or by confining the telomere to specific sub- checkpoint PIKK proteins become only transiently acti-

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Figure 3. Schematic representation of the mechanisms controlling telomere length homeostasis, telomere elongation of a short telomere, and the generation of a DDR at a critically short telomere. At a telomere in equilibrium, telomere factors (TFs) inhibit the activation of upstream DNA damage kinases (PIKKs), preventing them from activating proteins (such as Cdc13p) that can trigger telomere elongation. PIKK activation is also inhibited by proteins (e.g., Cdc13p) that can restrain telomere resection and the conse- quent accumulation of RPA on telomeric DNA. Telomere shortening causes the loss of telomere bound TFs, resulting in diminished PIKK inhibition and unleashing telomere elongation mechanisms. In the absence of telomere maintenance mechanisms, further telomere shortening leads to the loss of factors such as Cdc13p that prevent single-stranded DNA erosion, leading to unrestrained resection; this may cause the generation of a single-stranded DNA/RPA complex of a sufficient length to trigger the generation of a robust DDR. vated at telomeres at the end of S phase and that this functional telomeres: it recognizes both structures and is activation is coupled to effective telomere end-mainte- active at both (Fig. 3). However, whereas its activity at nance. Indeed, regulating telomerase access by such a functional telomeres is restrained by telomere-bound mechanism might provide opportunities for the cell to factors and thereby channelled towards telomere homeo- target this enzyme most effectively to the shorter telo- stasis, at dysfunctional telomeres the DDR is unre- meres in the population that are in most need of length- strained and enforces a DNA damage checkpoint involv- ening. Although such a control mechanism is hypotheti- ing the entire cascade of DDR factors. cal, we note that yeast Cdc13p has several conserved Future directions PIKK consensus phosphorylation sites, raising the possi- bility that such phosphorylations control the ability of Over the past decade, there has been much progress to- the Cdc13p complex to recruit telomerase and/or cap wards understanding the normal structure and functions telomeric ends (DuBois et al. 2002). A variation on the of telomeres, how telomere homeostasis is maintained, above models is one in which telomeric-binding factors and how telomeres are prevented from being recognized modulate the activity of PIKKs by allowing the PIKKs to as DNA damage. Strikingly, this work has revealed that phosphorylate proteins involved in normal telomere ho- normal telomere maintenance requires many proteins meostasis but preventing them from acting on down- associated with the DDR. Although we now have some stream components and generating a full-blown DDR. knowledge of how these proteins function, there is still One way this could be achieved is through differential much to be learned. One of the most important issues use of the mediators of the pathway; indeed, mediators facing the telomere field is to define precisely what it is such as BRCA1 are only required for the phosphorylation about telomeres that prevent them from being recog- of a subset of ATM and ATR substrates (Foray et al. nized as DNA damage. Many insights into this will 2003), suggesting that different components of the DDR surely come from further defining the structure and pathway are required to differing extents depending on functions of telomere-bound proteins and by establish- the initial signal and the final outcome. ing the range of proteins targeted by the checkpoint In summary, we propose that the DDR apparatus in PIKKs, both at the telomere and in the DDR. Another fact does not distinguish between functional and dys- major challenge will be to establish how the access of

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d’Adda di Fagagna et al. telomerase to the telomere is tightly controlled. In addi- Hekimi, S. 2001. The C. elegans maternal-effect gene clk-2 is tion, it will be interesting to ascertain whether addi- essential for embryonic development, encodes a protein ho- tional DDR proteins act at normal and/or dysfunctional mologous to yeast Tel2p and affects telomere length. Devel- telomeres. Such work is likely to provide insights not opment 128: 4045–4055. only into telomere biology but also into responses to Bertuch, A.A. and Lundblad, V. 2003. The Ku heterodimer per- forms separable activities at double-strand breaks and chro- DNA damage in a wider context. In this regard, it will mosome termini. Mol. Cell. Biol. 23: 8202–8215. also be of great interest to see whether further factors Blackburn, E.H. 2000. Telomere states and cell fates. Nature that were initially identified through their functions at 408: 53–56. the telomere actually have more widespread roles in the Blasco, M.A. 2002. Mouse models to study the role of telomeres DDR. Finally, it will be of key importance to establish in cancer, aging and DNA repair. Eur. J. Cancer 38: 2222– how deregulation of pathways of telomere maintenance 2228. can lead to cancer and, perhaps, also contributes to a Bomgarden, R.D., Yean, D., Yee, M.C., and Cimprich, K.A. range of other age-related pathologies. Given the intense 2004. A novel protein activity mediates DNA binding of an activity taking place in the telomere and DDR fields, it ATR–ATRIP complex. J. Biol. Chem. 279: 13346–13353. seems safe to predict that the answers to these and other Booth, C., Griffith, E., Brady, G., and Lydall, D. 2001. Quanti- tative amplification of single-stranded DNA (QAOS) demon- questions will soon be upon us. strates that cdc13-1 mutants generate ssDNA in a telomere to centromere direction. Nucleic Acids Res. 29: 4414–4422. Boulton, S.J. and Jackson, S.P. 1996. Identification of a Saccha- Acknowledgments romyces cerevisiae Ku80 homologue: Roles in DNA double- We apologize to those whose work was not cited because of strand break rejoining and in telomeric maintenance. space restrictions on this article. We thank K. Dry for editorial Nucleic Acids Res. 24: 4639–4648. assistance and A. Decottignies, M. Foiani, M.P. Longhese, D. ———. 1998. Components of the Ku-dependent non-homolo- Lydall, J. Karlseder, S. Giavara, S. Gravel, and P. Reaper for their gous end-joining pathway are involved in telomeric length constructive comments. The Jackson laboratory is funded by maintenance and telomeric silencing. EMBO J. 17: 1819– grants from Cancer Research UK, the European Union, and the 1828. AT Medical Research Trust. S.H.T. was supported by a Royal Brown, E.J. and Baltimore, D. 2000. ATR disruption leads to Society Dorothy Hodgkin Fellowship. F.dA.dF. is supported by chromosomal fragmentation and early embryonic lethality. AIRC (Associazione Italiana Ricerca sul Cancro). Genes & Dev. 14: 397–402. Bundock, P. and Hooykaas, P. 2002. Severe developmental de- fects, hypersensitivity to DNA-damaging agents, and length- References ened telomeres in Arabidopsis MRE11 mutants. Plant Cell 14: 2451–2462. Adams, I.R. and McLaren, A. 2004. Identification and charac- Bundock, P., van Attikum, H., and Hooykaas, P. 2002. Increased terisation of mRif1: A mouse telomere-associated protein telomere length and hypersensitivity to DNA damaging highly expressed in germ cells and embryo-derived pluripo- agents in an Arabidopsis KU70 mutant. Nucleic Acids Res. tent stem cells. Dev. Dyn. 229: 733–744. 30: 3395–3400. Ahmed, S. and Hodgkin, J. 2000. MRT-2 checkpoint protein is Chahwan, C., Nakamura, T.M., Sivakumar, S., Russell, P., and required for germline immortality and telomere replication Rhind, N. 2003. The fission yeast Rad32 (Mre11)–Rad50– in C-elegans. Nature 403: 159–164. Nbs1 complex is required for the S-phase DNA damage Ahmed, S., Alpi, A., Hengartner, M.O., and Gartner, A. 2001. C. checkpoint. Mol. Cell. Biol. 23: 6564–6573. elegans RAD-5/CLK-2 defines a new DNA damage check- Chakhparonian, M. and Wellinger, R.J. 2003. Telomere mainte- point protein. Curr. Biol. 11: 1934–1944. nance and DNA replication: How closely are these two con- Bailey, S.M., Cornforth, M.N., Kurimasa, A., Chen, D.J., and nected? Trends Genet. 19: 439–446. Goodwin E.H. 2001. Strand-specific postreplicative process- Chan, S.W.L., Chang, J., Prescott, J., and Blackburn, E.H. 2001. ing of mammalian telomeres. Science 293: 2462–2465. Altering telomere structure allows telomerase to act in yeast Bailey, S.M., Brenneman, M.A., Halbrook, J., Nickoloff, J.A., lacking ATM kinases. Curr. Biol. 11: 1240–1250. Ullrich, R.L., and Goodwin, E.H. 2004. The kinase activity of Chen, Q., Ijpma, A., and Greider, C.W. 2001. Two survivor path- DNA-PK is required to protect mammalian telomeres. DNA ways that allow growth in the absence of telomerase are Repair 3: 225–233. generated by distinct telomere recombination events. Mol. Bakkenist, C.J. and Kastan, M.B. 2003. DNA damage activates Cell. Biol. 21: 1819–1827. ATM through intermolecular autophosphorylation and Chin, L., Artandi, S.E., Shen, Q., Tam, A., Lee, S.-L., Gottlieb, dimer dissociation. Nature 421: 499–506. G.J., Greider, C.W., and DePinho, R.A. 1999. p53 deficiency Bakkenist, C.J., Drissi, R., Wu, J., Kastan, M.B., and Dome, J.S. rescues the adverse effects of telomere loss and cooperates 2004. Disappearance of the telomere dysfunction-induced with telomere dysfunction to accelerate carcinogenesis. Cell stress response in fully senescent cells. Cancer Res. 97: 527–538. 64: 3748–3752. Colgin, L.M., Baran, K., Baumann, P., Cech, T.R., and Reddel, Bartek, J. and Lukas, J. 2003. Chk1 and Chk2 kinases in check- R.R. 2003. Human POT1 facilitates telomere elongation by point control and cancer. Cancer Cell 3: 421–429. telomerase. Curr. Biol. 13: 942–946. Baumann, P. and Cech, T.R. 2000. Protection of telomeres by Corda, Y., Schramke, V., Longhese, M.P., Smokvina, T., Pac- the Ku protein in fission yeast. Mol. Biol. Cell 11: 3265– iotti, V., Brevet, V., Gilson, E., and Geli, V. 1999. Interaction 3275. between Set1p and checkpoint protein Mec3p in DNA repair ———. 2001. Pot1, the putative telomere end-binding protein in and telomere functions. Nat. Genet. 21: 204–208. fission yeast and humans. Science 292: 1171–1175. Cosgrove, A.J., Nieduszynski, C.A., and Donaldson, A.D. 2002. Benard, C., McCright, B., Zhang, Y., Felkai, S., Lakowski, B., and Ku complex controls the replication time of DNA in telo-

1794 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press

Telomeres and the DNA-damage response

mere regions. Genes & Dev. 16: 2485–2490. mediates chromosomal fusions and apoptosis caused by Craven, R.J. and Petes, T.D. 1999. Dependence of the regulation critically short telomeres. EMBO J. 21: 2207–2219. of telomere length on the type of subtelomeric repeat in the Espejel, S., Franco, S., Sgura, A., Gae, D., Bailey, S.M., Taccioli, yeast Saccharomyces cerevisiae. Genetics 152: 1531–1541. G.E., and Blasco, M.A. 2002b. Functional interaction be- d’Adda di Fagagna, F., Hande, M.P., Tong, W.-M., Lansdorp, tween DNA-PKcs and telomerase in telomere length main- P.M., Wang, Z.-Q., and Jackson, S.P. 1999. Functions of poly- tenance. EMBO J. 21: 6275–6287. (ADP-ribose) polymerase in controlling telomere length and Fernandez-Capetillo, O. and Nussenzweig, A. 2004. Linking chromosomal stability. Nat. Genet. 23: 76–80. histone deacetylation with the repair of DNA breaks. Proc. d’Adda di Fagagna, F., Hande, M.P., Tong, W.-M., Roth, D., Natl Acad Sci 101: 1427–1428. Lansdorp, P.M., Wang, Z.-Q., and Jackson, S.P. 2001. Effects Fernandez-Capetillo, O., Liebe, B., Scherthan, H., and Nussen- of DNA nonhomologous end-joining factors on telomere zweig, A. 2003. H2AX regulates meiotic telomere clustering. length and chromosomal stability in mammalian cells. Curr. J. Cell Biol. 163: 15–20. Biol. 11: 1192–1196. Ferreira, M.G. and Cooper, J.P. 2001. The fission yeast Taz1 d’Adda di Fagagna, F., Reaper, P.M., Clay-Farrace, L., Fiegler, H., protein protects chromosomes from Ku-dependent end-to- Carr, P., Von Zglinicki, T., Saretzki, G., Carter, N.P., and end fusions. Mol. Cell 7: 55–63. Jackson S.P. 2003. A DNA damage checkpoint response in Figueiredo, L.M., Freitas-Junior, L.H., Bottius, E., Olivo-Marin, telomere-initiated senescence. Nature 426: 194–198. J.C., and Scherf, A. 2002. A central role for Plasmodium fal- Dahlen, M., Olsson, T., Kanter-Smoler, G., Ramne, A., and Sun- ciparum subtelomeric regions in spatial positioning and telo- nerhagen, P. 1998. Regulation of telomere length by check- mere length regulation. EMBO J. 21: 815–824. point genes in Schizosaccharomyces pombe. Mol. Biol. Cell Foray, N., Marot, D., Gabriel, A., Randrianarison, V., Carr, 9: 611–621. A.M., Perricaudet, M., Ashworth, A., and Jeggo, P. 2003. A D’Amours, D. and Jackson, S.P. 2001. The yeast Xrs2 complex subset of ATM- and ATR-dependent phosphorylation events functions in S phase checkpoint regulation. Genes & Dev. requires the BRCA1 protein. EMBO J. 22: 2860–2871. 15: 2238–2249. Friedberg, E.C., Walker, G.C., and Siede, W. 1995. DNA repair ———. 2002. The Mre11 complex: At the crossroads of DNA and mutagenesis. ASM Press, Washington, D.C. repair and checkpoint signalling. Nat. Rev. Mol. Cell Biol. Gallego, M.E. and White, C.I. 2001. RAD50 function is essential 3: 317–327. for telomere maintenance in Arabidopsis. Proc. Nat. Acad. D’Amours, D., Desnoyers, S., D’Silva, I., and Poirier, G.G. 1999. Sci. 98: 1711–1716. Poly(ADP-ribosyl)ation reactions in the regulation of Garvik, B., Carson, M., and Hartwell, L. 1995. Single-stranded nuclear functions. Biochem. J. 342: 249–268. DNA arising at telomeres in cdc13 mutants may constitute de Bruin, D., Zaman, Z., Liberatore, R.A., and Ptashne, M. 2001. a specific signal for the RAD9 checkpoint. Mol. Cell. Biol. Telomere looping permits gene activation by a downstream 15: 6128–6138. UAS in yeast. Nature 409: 109–113. Gilbert, C.S., Green, C.M., and Lowndes, N.F. 2001. Budding de Laat, W.L., Jaspers, N.G.J., and Hoeijmakers, J.H.J. 1999. Mo- yeast Rad9 is an ATP-dependent Rad53 activating machine. lecular mechanism of nucleotide excision repair. Genes & Mol. Cell 8: 129–136. Dev. 13: 768–785. Gilley, D., Tanaka, H., Hande, M.P., Kurimasa, A., Li, G.C., and Diede, S.J. and Gottschling, D.E. 1999. Telomerase-mediated Chen, D.J. 2001. DNA-PKcs is critical for telomere capping. telomere addition in vivo requires DNA primase and DNA Proc. Nat. Acad. Sci. 98: 15084–15088. polymerases ␣ and ␦. Cell 99: 723–733. Goldberg, M., Stucki, M., Falck, J., D’Amours, D., Rahman, D., ———. 2001. Exonuclease activity is required for sequence ad- Pappin, D., Bartek, J., and Jackson, S.P. 2003. MDC1 is re- dition and Cdc13p loading at a de novo telomere. Curr. Biol. quired for the intra-S-phase DNA damage checkpoint. Na- 11: 1336–1340. ture 421: 952–956. Downs, J.A. and Jackson, S.P. 2004. A means to a DNA end: The Gotta, M., Laroche, T., Formenton, A., Maillet, L., Scherthan, many roles of Ku. Nat. Rev. Mol. Cell Biol. 5: 367–378. H., and Gasser, S.M. 1996. The clustering of telomeres and Downs, J.A., Kosmidou, E., Morgan, A., and Jackson, S.P. 2003. colocalization with Rap1, Sir3, and Sir4 proteins in wild type Suppression of homologous recombination by the Saccharo- Saccharomyces cerevisiae. J. Cell Biol. 134: 1349–1363. myces cerevisiae linker histone. Mol. Cell 11: 1685–1692. Goytisolo, F.A., Samper, E., Martin-Caballero, J., Finnon, P., Driller, L., Wellinger, R.J., Larrivee, M., Kremmer, E., Jaklin, S., Herrera, E., Flores, J.M., Bouffler, S.D., and Blasco, M.A. and Feldmann, H.M. 2000. A short C-terminal domain of 2000. Short telomeres result in organismal hypersensitivity Yku70p is essential for telomere maintenance. J. Biol. Chem. to ionizing radiation in mammals. J. Exp. Med. 192: 1625– 275: 24921–24927. 1636. DuBois, M.L., Haimberger, Z.W., McIntosh, M.W., and Grandin, N. and Charbonneau, M. 2003. The Rad51 pathway of Gottschling, D.E. 2002. A quantitative assay for telomere telomerase-independent maintenance of telomeres can am- protection in Saccharomyces cerevisiae. Genetics 161: 995– plify TG1–3 sequences in yku and cdc13 mutants of Saccha- 1013. romyces cerevisiae. Mol. Cell. Biol. 23: 3721–3734. Dunham, M.A., Neumann, A.A., Fasching, C.L., and Reddel, Grandin, N., Reed, S.I., and Charbonneau, M. 1997. Stn1, a new R.R. 2000. Telomere maintenance by recombination in hu- Saccharomyces cerevisiae protein, is implicated in telomere man cells. Nature Genet. 26: 447–450. size regulation in association with Cdc13. Genes & Dev. Enomoto, S., Glowczewski, L., and Berman, J. 2002. MEC3, 11: 512–527. MEC1, and DDC2 are essential components of a telomere Grandin, N., Damon, C., and Charbonneau, M. 2001a. Cdc13 checkpoint pathway required for cell cycle arrest during se- prevents telomere uncapping and Rad50-dependent homolo- nescence in Saccharomyces cerevisiae. Mol. Biol. Cell gous recombination. EMBO J. 20: 6127–6139. 13: 2626–2638. ———. 2001b. Ten1 functions in telomere end protection and Espejel, S., Franco, S., Rodríguez-Perales, S., Bouffler, S.D., length regulation in association with Stn1 and Cdc13. Cigudosa, J.C., and Blasco, M.A. 2002a. Mammalian Ku86 EMBO J. 20: 1173–1183.

GENES & DEVELOPMENT 1795 Downloaded from genesdev.cshlp.org on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press

d’Adda di Fagagna et al.

Gravel, S. and Wellinger, R.J. 2002. Maintenance of double- Karlseder, J., Broccoli, D., Dai, Y., Hardy, S., and de Lange, T. stranded telomeric repeats as the critical determinant for 1999. p53- and ATM-dependent apoptosis induced by telo- cell viability in yeast cells lacking Ku. Mol. Cell. Biol. meres lacking TRF2. Science 283: 1321–1325. 22: 2182–2193. Karlseder, J., Smogorzewska, A., and de Lange, T. 2002. Senes- Gravel, S., Larrivée, M., Labrecque, P., and Wellinger, R.J. 1998. cence induced by altered telomere state, not telomere loss. Yeast Ku as a regulator of chromosomal DNA end structure. Science 295: 2446–2449. Science 280: 741–744. Karnitz, L.M. 2004. Dial 9–1–1 for DNA damage: The Rad9– Greenwell, P.W., Kronmal, S.L., Porter, S.E., Gassenhuber, J., Hus1–Rad1 clamp complex. DNA Repair (Amst.) (in press). Obermaier, B., and Petes, T.D. 1995. TEL1, a gene involved Khanna, K.K. and Jackson, S.P. 2001. DNA double-strand in controlling telomere length in S. cerevisiae, is homolo- breaks: Signaling, repair and the cancer connection. Nat. gous to the human ataxia telangiectasia gene. Cell 82: 823– Genet. 27: 247–254. 829. Kharbanda, S., Kumar, V., Dhar, S., Pandey, P., Chen, C., Grenon, M., Gilbert, C., and Lowndes, N.F. 2001. Checkpoint Majumder, P., Yuan, Z.-M., Whang, Y., Strauss, W., Pandita, activation in response to double-strand breaks requires the T.K., et al. 2000. Regulation of the hTERT telomerase cata- Mre11/Rad50/Xrs2 complex. Nat. Cell Biol. 3: 844–847. lytic subunit by the c-Abl tyrosine kinase. Curr. Biol. Griffith, J.D., Comeau, L., Rosenfield, S., Stansel, R.M., Bianchi, 10: 568–575. A., Moss, H., and de Lange, T. 1999. Mammalian telomeres Kota, R.S. and Runge, K.W. 1998. The yeast telomere length end in a large duplex loop. Cell 97: 503–514. regulator TEL2 encodes a protein that binds to telomeric Hande, M.P., Balajee, A.S., and Natarajan, A.T. 1997. Induction DNA. Nucleic Acids Res. 26: 1528–1535. of telomerase activity by UV-irradiation in Chinese hamster Laroche, T., Martin, S.G., Gotta, M., Gorham, H.C., Pryde, F.E., cells. Oncogene 15: 1747–1752. Louis, E.J., and Gasser, S.M. 1998. Mutation of yeast Ku Hande, M.P., Lansdorp, P.M., and Natarajan, A.T. 1998. Induc- genes disrupts the subnuclear organization of telomeres. tion of telomerase activity by in vivo X-irradiation of mouse Curr. Biol. 8: 653–656. splenocytes and its possible role in chromosome healing. Larrivée, M., Lebel, C., and Wellinger, R.J. 2004. The generation Mutat. Res. 404: 205–214. of proper constitutive G-tails on yeast telomeres is depen- Hande, M.P., Balajee, A.S., Tchirkov, A., Wynshaw-Boris, A., dent on the MRX-complex. Genes & Dev. 18: 1391–1396. and Lansdorp, P.M. 2001. Extra-chromosomal telomeric Le, S., Moore, J.K., Haber, J.E., and Greider, C.W. 1999. RAD50 DNA in cells from Atm−/− mice and patients with ataxia- and RAD51 define two pathways that collaborate to main- telangiectasia. Human Mol. Genet. 10: 519–528. tain telomeres in the absence of telomerase. Genetics Hartsuiker, E., Vaessen, E., Carr, A.M., and Kohli, J. 2001. Fis- 152: 143–152. sion yeast Rad50 stimulates sister chromatid recombination Lei, M., Podell, E.R., Baumann, P., and Cech, T.R. 2003. DNA and links cohesion with repair. EMBO J. 20: 6660–6671. self-recognition in the structure of Pot1 bound to telomeric Henson, J.D., Neumann, A.A., Yeager, T.R., and Reddel, R.R. single-stranded DNA. Nature 426: 198–203. 2002. Alternative lengthening of telomeres in mammalian Li, B. and de Lange, T. 2003. Rap1 affects the length and het- cells. Oncogene 21: 598–610. erogeneity of human telomeres. Mol. Biol. Cell 14: 5060– Herbig, U., Jobling, W.A., Chen, B.P., Chen, D.J., and Sedivy, 5068. J.M. 2004. Telomere shortening triggers senescence of hu- Li, G., Nelsen, C., and Hendrickson, E.A. 2002. Ku86 is essen- man cells through a pathway involving ATM, p53, and tial in human somatic cells. Proc. Natl. Acad. Sci. 99: 832– p21(CIP1), but not p16(INK4a). Mol. Cell 14: 501–513. 837. Herrmann, G., Lindahl, T., and Schar, P. 1998. Saccharomyces Lieber, M.R., Ma, Y., Pannicke, U., and Schwarz, K. 2003. cerevisiae LIF1: A function involved in DNA double-strand Mechanism and regulation of human non-homologous DNA break repair related to mammalian XRCC4. EMBO J. end-joining. Nat. Rev. Mol. Cell. Biol. 4: 712–720. 17: 4188–4198. Lim, C.-S., Mian, I.S., Dernburg, A.F., and Campisi, J. 2001. C. Hsu, H.-L., Gilley, D., Blackburn, E.H., and Chen, D.J. 1999. Ku elegans clk-2, a gene that limits life span, encodes a telomere is associated with the telomere in mammals. Proc. Natl. length regulator similar to yeast telomere binding protein Acad. Sci. 96: 12454–12458. Tel2p. Curr. Biol. 11: 1706–1710. Hsu, H.-L., Gilley, D., Galande, S.A., Hande, M.P., Allen, B., Linke, S.P., Sengupta, S., Khabie, N., Jeffries, B.A., Buchhop, S., Kim, S.-H., Li, G.C., Campisi, J., Kohwi-Shigematsu, T., and Miska, S., Henning, W., Pedeux, R., Wang, X.W., Hofseth, Chen, D.J. 2000. Ku acts in a unique way at the mammalian L.J., et al. 2003. p53 interacts with hRAD51 and hRAD54, telomere to prevent end joining. Genes & Dev. 14: 2807– and directly modulates homologous recombination. Cancer 2812. Res. 63: 2596–2605. IJpma, A.S. and Greider, C.W. 2003. Short telomeres induce a Liti, G. and Louis, E.J. 2003. NEJ1 prevents NHEJ-dependent DNA damage response in Saccharomyces cerevisiae. Mol. telomere fusions in yeast without telomerase. Mol. Cell Biol. Cell 14: 987–1001. 11: 1373–1378. Jaco, I., Munoz, P., Goytisolo, F., Wesoly, J., Bailey, S., Taccioli, Loayza, D. and de Lange, T. 2003. POT1 as a terminal trans- G., and Blasco, M.A. 2003. Role of mammalian Rad54 in ducer of TRF1 telomere length control. Nature 424: 1013– telomere length maintenance. Mol. Cell. Biol. 23: 5572– 1018. 5580. Longhese, M.P., Paciotti, V., Neecke, H., and Lucchini, G. 2000. Jiang, N., Benard, C.Y., Kebir, H., Shoubridge, E.A., and Hekimi, Checkpoint proteins influence telomeric silencing and S. 2003. Human CLK2 links cell cycle progression, apo- length maintenance in budding yeast. Genetics 155: 1577– ptosis, and telomere length regulation. J. Biol. Chem. 1591. 278: 21678–21684. Lundblad, V. 2002. Telomere maintenance without telomerase. Kanellis, P., Agyei, R., and Durocher, D. 2003. Elg1 forms an Oncogene 21: 522–531. alternative PCNA-interacting RFC complex required to Lundblad, V. and Blackburn, E.H. 1993. An alternative pathway maintain genome stability. Curr. Biol. 13: 1583–1595. for yeast telomere maintenance rescues est1− senescence.

1796 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press

Telomeres and the DNA-damage response

Cell 73: 347–360. 5059. Lustig, A.J. and Petes, T.D. 1986. Identification of yeast mutants Naito, T., Matsuura, A., and Ishikawa, F. 1998. Circular chro- with altered telomere structure. Proc. Natl. Acad. Sci. mosome formation in a fission yeast mutant defective in two 83: 1398–1402. ATM homologues. Nat. Genet. 20: 203–206. Lydall, D. and Weinert, T. 1995. Yeast checkpoint genes in Nakamura, T.M., Cooper, J.P., and Cech, T.R. 1998. Two modes DNA damage processing: Implications for repair and arrest. of survival of fission yeast without telomerase. Science Science 270: 1488–1491. 282: 493–496. Mallory, J.C. and Petes, T.D. 2000. Protein kinase activity of Nakamura, T.M., Moser, B.A., and Russell, P. 2002. Telomere Tel1p and Mec1p, two Saccharomyces cerevisiae proteins binding of checkpoint sensor and DNA repair proteins con- related to the human ATM protein kinase. Proc. Natl. Acad. tributes to maintenance of functional fission yeast telo- Sci. 97: 13749–13754. meres. Genetics 161: 1437–1452. Mallory, J.C., Bashkirov, V.I., Trujillo, K.M., Solinger, J.A., Nautiyal, S., DeRisi, J.L., and Blackburn, E.H. 2002. The ge- Dominska, M., Sung, P., Heyer, W.D., and Petes, T.D. 2003. nome-wide expression response to telomerase deletion in Amino acid changes in Xrs2p, Dun1p, and Rfa2p that remove Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. 99: 9316– the preferred targets of the ATM family of protein kinases do 9321. not affect DNA repair or telomere length in Saccharomyces Neumann, A.A. and Reddel, R.R. 2002. Telomere maintenance cerevisiae. DNA Repair (Amst.) 2: 1041–1064. and cancer—Look, no telomerase. Nat. Rev. Cancer 2: 879– Manolis, K.G., Nimmo, E.R., Hartsuiker, E., Carr, A.M., Jeggo, 884. P.A., and Allshire, R.C. 2001. Novel functional require- Niida, H., Shinkai, Y., Hande, M.P., Matsumoto, T., Takehara, ments for non-homologous DNA end joining in Schizosac- S., Tachibana, M., Oshimura, M., Lansdorp, P.M., and Furui- charomyces pombe. EMBO J. 20: 210–221. chi, Y. 2000. Telomere maintenance in telomerase-deficient Marcand, S., Gilson, E., and Shore, D. 1997. A protein-counting mouse embryonic stem cells: Characterization of an ampli- mechanism for telomere length regulation in yeast. Science fied telomeric DNA. Mol. Cell. Biol. 20: 4115–4127. 275: 986–990. Nugent, C.I., Bosco, G., Ross, L.O., Evans, S.K., Salinger, A.P., Maringele, L. and Lydall, D. 2002. EXO1-dependent single- Moore, J.K., Haber, J.E., and Lundblad, V. 1998. Telomere stranded DNA at telomeres activates subsets of DNA dam- maintenance is dependent on activities required for end re- age and spindle checkpoint pathways in budding yeast pair of double-strand breaks. Curr. Biol. 8: 657–660. yku70⌬ mutants. Genes & Dev. 16: 1919–1933. Nussenzweig, A. 2004. H2AX: The histone guardian of the ge- Matsuura, A., Naito, T., and Ishikawa, F. 1999. Genetic control nome. DNA Repair (Amst.) (in press). of telomere integrity in Schizosaccharomyces pombe: rad3+ Parenteau, J. and Wellinger, R.J. 2002. Differential processing of and tel1+ are parts of two regulatory networks independent leading- and lagging-strand ends at Saccharomyces cerevi- of the downstream protein kinases chk1+ and cds1+. Genet- siae telomeres revealed by the absence of Rad27p nuclease. ics 152: 1501–1512. Genetics 162: 1583–1594. McEachern, M.J., Krauskopf, A., and Blackburn, E.H. 2000. Pellicioli, A., Lee, S.B., Lucca, C., Foiani, M., and Haber, J.E. Telomeres and their control. Annu. Rev. Genet. 34: 331– 2001. Regulation of Saccharomyces Rad53 checkpoint ki- 358. nase during adaptation from DNA damage-induced G2/M Mekeel, K.L., Tang, W., Kachnic, L.A., Luo, C.-M., DeFrank, arrest. Mol. Cell 7: 293–300. J.S., and Powell, S.N. 1997. Inactivation of p53 results Pennock, E., Buckley, K., and Lundblad, V. 2001. Cdc13 delivers in high rates of homologous recombination. Oncogene separate complexes to the telomere for end protection and 14: 1847–1857. replication. Cell 104: 387–396. Metcalfe, J.A., Parkhill, J., Campbell, L., Stacey, M., Biggs, P., Peterson, S.E., Stellwagen, A.E., Diede, S.J., Singer, M.S., Haim- Byrd, P.J., and Taylor, A.M.R. 1996. Accelerated telomere berger, Z.W., Johnson, C.O., Tzoneva, M., and Gottschling, shortening in ataxia telangiectasia. Nat. Genet. 13: 350–353. D.E. 2001. The function of a stem-loop in telomerase RNA is Mieczkowski, P.A., Mieczkowska, J.O., Dominska, M., and linked to the DNA repair protein Ku. Nat. Genet. 27: 64–67. Petes T.D. 2003. Genetic regulation of telomere–telomere Polotnianka, R.M., Li, J., and Lustig, A.J. 1998. The yeast Ku fusions in the yeast Saccharomyces cerevisae. Proc. Natl. heterodimer is essential for protection of the telomere Acad. Sci. 100: 10854–10859. against nucleolytic and recombinational activities. Curr. Mitton-Fry, R.M., Anderson, E.M., Hughes, T.R., Lundblad, V., Biol. 8: 831–834. and Wuttke, D.S. 2002. Conserved structure for single- Porter, S.E., Greenwell, P.W., Ritchie, K.B., and Petes, T.D. stranded telomeric DNA recognition. Science 296: 145–147. 1996. The DNA-binding protein Hdf1p (a putative Ku homo- Moreau, S., Ferguson, J.R., and Symington, L.S. 1999. The nucle- log) is required for maintaining normal telomere length in ase activity of Mre11 is required for meiosis but not for mat- Saccharomyces cerevisiae. Nucleic Acids Res. 24: 582–585. ing type switching, end joining, or telomere maintenance. Ranganathan, V., Heine, W.F., Ciccone, D.N., Rudolph, K.L., Mol. Cell. Biol. 19: 556–566. Wu, X., Chang, S., Hai, H., Ahearn, I.M., Livingston, D.M., Munoz-Jordan, J.L., Cross, G.A.M., de Lange, T., and Griffith, Resnick, I., et al. 2001. Rescue of a telomere length defect of J.D. 2001. T-loops at trypanosome telomeres. EMBO J. Nijmegen breakage syndrome cells requires NBS and telom- 20: 579–588. erase catalytic subunit. Curr. Biol. 11: 962–966. Myung, K., He, D.M., Lee, S.E., and Hendrickson, E.A. 1997. Ray, A. and Runge, K.W. 1999. Varying the number of telomere- KARP-1: A novel leucine zipper protein expressed from the bound proteins does not alter telomere length in tel1⌬ cells. Ku86 autoantigen locus is implicated in the control of DNA- Proc. Natl. Acad. Sci. 96: 15044–15049. dependent protein kinase activity. EMBO J. 16: 3172–3184. Reichenbach, P., Höss, M., Azzalin, C.M., Nabholz, M., Bucher, Myung, K., Ghosh, G., Fattah, F.J., Li, G., Kim, H., Dutia, A., P., and Lingner, J. 2003. A human homolog of yeast est1 Pak, E., Smith, S., and Hendrickson, E.A. 2004. Regulation of associates with telomerase and uncaps chromosome ends telomere length and suppression of genomic instability in when overexpressed. Curr. Biol. 13: 568–574. human somatic cells by Ku86. Mol. Cell. Biol. 24: 5050– Rich, T., Allen, R.L., and Wyllie, A.H. 2000. Defying death after

GENES & DEVELOPMENT 1797 Downloaded from genesdev.cshlp.org on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press

d’Adda di Fagagna et al.

DNA damage. Nature 407: 777–783. protein kinase. Genes & Dev. 13: 916–934. Riha, K. and Shippen, D.E. 2003. Ku is required for telomeric Smith, J., Zou, H., and Rothstein, R. 2000. Characterization of C-rich strand maintenance but not for end-to-end chromo- genetic interactions with RFA1: The role of RPA in DNA some fusions in Arabidopsis. Proc. Natl. Acad. Sci. 100: 611– replication and telomere maintenance. Biochimie 82: 71–78. 615. Smogorzewska, A., Karlseder, J., Holtgreve-Grez, H., Jauch, A., Riha, K., McKnight, T.D., Griffing, L.R., and Shippen, D.E. and de Lange, T. 2002. DNA ligase IV-dependent NHEJ of 2001. Living with genome instability: Plant responses to deprotected mammalian telomeres in G1 and G2. Curr. Biol. telomere dysfunction. Science 291: 1797–1800. 12: 1635. Riha, K., Watson, J.M., Parkey, J., and Shippen, D.E. 2002. Telo- Smolikov, S., Mazor, Y., and Krauskopf, A. 2004. ELG1, a regu- mere length deregulation and enhanced sensitivity to geno- lator of genome stability, has a role in telomere length regu- toxic stress in Arabidopsis mutants deficient in Ku70. lation and in silencing. Proc. Natl. Acad. Sci. 101: 1656– EMBO J. 21: 2819–2826. 1661. Ritchie, K.B. and Petes, T.D. 2000. The Mre11p/Rad50p/Xrs2p Snow, B.E., Erdmann, N., Cruickshank, J., Goldman, H., Gill complex and the Tel1p function in a single pathway for telo- R.M., Robinson, M.O., and Harrington, L. 2003. Functional mere maintenance in yeast. Genetics 155: 475–479. conservation of the telomerase protein est1p in humans. Ritchie, K.B., Mallory, J.C., and Petes, T.D. 1999. Interactions of Curr. Biol. 13: 698–704. TLC1 (which encodes the RNA subunit of telomerase), Song, K., Jung, D., Jung, Y., Lee, S.-G., and Lee, I. 2000. Inter- TEL1, and MEC1 in regulating telomere length in the yeast action of human Ku70 with TRF2. FEBS Lett. 481: 81–85. Saccharomyces cerevisiae. Mol. Cell. Biol. 19: 6065–6075. Stansel, R.M., de Lange, T., and Griffith, J.D. 2001. T-loop as- Rouse, J. and Jackson, S.P. 2002a. Interfaces between the de- sembly in vitro involves binding of TRF2 near the 3Ј telo- tection, signaling, and repair of DNA damage. Science meric overhang. EMBO J. 20: 5532–5540. 297: 547–551. Stavenhagen, J.B. and Zakian, V.A. 1998. Yeast telomeres exert ———. 2002b. Lcd1p recruits Mec1p to DNA lesions in vitro a position effect on recombination between internal tracts of and in vivo. Mol. Cell 9: 857–869. yeast telomeric DNA. Genes & Dev. 12: 3044–3058. Roy, R., Meier, B., McAinsh, A.D., Feldmann, H.M., and Jack- Stellwagen, A.E., Haimberger, Z.W., Veatch, J.R., and son, S.P. 2004. Separation-of-function mutants of yeast Ku80 Gottschling, D.E. 2003. Ku interacts with telomerase RNA reveal a Yku80p–Sir4p interaction involved in telomeric si- to promote telomere addition at native and broken chromo- lencing. J. Biol. Chem. 279: 86–94. some ends. Genes & Dev. 17: 2384–2395. Runge, K.W. and Zakian, V.A. 1996. TEL2,anessentialgene Stucki, M. and Jackson, S.P. 2004. MDC1/NFBD1: A key regu- required for telomere length regulation and telomere posi- lator of the DNA damage response in higher eukaryotes. tion effect in Saccharomyces cerevisiae. Mol. Cell. Biol. DNA Repair (Amst.) (in press). 16: 3094–3105. Sung, P., Krejci, L., Van Komen, S., and Sehorn, M.G. 2003. Samper, E., Goytisolo, F.A., Slijepcevic, P., van Buul, P.P.W., Rad51 recombinase and recombination mediators. J. Biol. and Blasco, M.A. 2000. Mammalian Ku86 protein prevents Chem. 278: 42729–42732. telomeric fusions independently of the length of TTAGGG Takai, H., Smogorzewska, A., and de Lange, T. 2003. DNA dam- repeats and the G-strand overhang. EMBO Rep. 1: 244–252. age foci at dysfunctional telomeres. Curr. Biol. 13: 1549– Samper, E., Goytisolo, F.A., Ménissier-de Murcia, J., González- 1556. Suarez, E., Cigudosa, J.C., de Murcia, G., and Blasco, M.A. Takata, H., Kanoh, Y., Gunge, N., Shirahige, K., and Matsuura, 2001. Normal telomere length and chromosomal end cap- A. 2004. Reciprocal association of the budding yeast ATM- ping in poly(ADP-ribose) polymerase-deficient mice and pri- related proteins Tel1 and Mec1 with telomeres in vivo. Mol. mary cells despite increased chromosomal instability. J. Cell Cell 14: 515–522. Biol. 154: 49–60. Tarsounas, M., Munoz, P., Claas, A., Smiraldo, P.G., Pittman, Scherthan, H. 2001. A bouquet makes ends meet. Nat. Rev. Mol. D.L., Blasco, M.A., and West, S.C. 2004. Telomere mainte- Cell. Biol. 2: 621–627. nance requires the RAD51D recombination/repair protein. Schmitt, C.A. 2003. Senescence, apoptosis and therapy—Cut- Cell 117: 337–347. ting the lifelines of cancer. Nat. Rev. Cancer 3: 286–295. Teng, S.C. and Zakian, V.A. 1999. Telomere–telomere recom- Schramke, V., Luciano, P., Brevet, V., Guillot, S., Corda, Y., bination is an efficient bypass pathway for telomere main- Longhese, M.P., Gilson, E., and Geli, V. 2004. RPA regulates tenance in Saccharomyces cerevisiae. Mol. Cell. Biol. telomerase action by providing Est1p access to chromosome 19: 8083–8093. ends. Nat. Genet. 36: 46–54. Teng, S.C., Chang, J., McCowan, B., and Zakian, V.A. 2000. Schultz, N., Lopez, E., Saleh-Gohari, N., and Helleday, T. 2003. Telomerase-independent lengthening of yeast telomeres oc- Poly(ADP-ribose) polymerase (PARP-1) has a controlling role curs by an abrupt Rad50p-dependent, Rif-inhibited recombi- in homologous recombination. Nucleic Acids Res. 31: 4959– national process. Mol. Cell 6: 947–952. 4964. Teo, S.-H. and Jackson, S.P. 1997. Identification of Saccharomy- Shiloh, Y. 2003. ATM and related protein kinases: Safeguarding ces cerevisiae DNA ligase IV: Involvement in DNA double- genome integrity. Nat. Rev. Cancer 3: 155–168. strand break repair. EMBO J. 16: 4788–4795. Shiomi, Y., Shinozaki, A., Nakada, D., Sugimoto, K., Usukura, Tong, W.-M., Hande, M.P., Lansdorp, P.M., and Wang, Z.-Q. J., Obuse, C., and Tsurimoto, T. 2002. Clamp and clamp 2001. DNA strand break-sensing molecule poly(ADP-ribose) loader structures of the human checkpoint protein com- polymerase cooperates with p53 in telomere function, chro- plexes, Rad9–1–1 and Rad17-RFC. Genes Cells 7: 861–868. mosome stability, and tumor suppression. Mol. Cell. Biol. Silverman, J., Takai, H., Buonomo, S.B.C., Eisenhaber, F., and de 21: 4046–4054. Lange, T. 2004. Human Rif1, ortholog of a yeast telomeric Tsai, Y.-L., Tseng, S.-F., Chang, S.-H., Lin, C.-C., and Teng, S.-C. protein, is regulated by ATM and 53BP1 and functions in the 2002. Involvement of replicative polymerases, Tel1p, S-phase checkpoint. Genes & Dev. (in press). Mec1p, Cdc13p, and the Ku complex in telomere–telomere Smith, G.C.M. and Jackson, S.P. 1999. The DNA-dependent recombination. Mol. Cell. Biol. 22: 5679–5687.

1798 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press

Telomeres and the DNA-damage response

Tsukamoto, Y., Kato, J., and Ikeda, H. 1997. Silencing factors Mol. Cell 12: 1489–1498. participate in DNA repair and recombination in Saccharo- Zou, L. and Elledge, S.J. 2003. Sensing DNA damage through myces cerevisiae. Nature 388: 900–903. ATRIP recognition of RPA–ssDNA complexes. Science Tsukamoto, Y., Taggart, A.K.P., and Zakian, V.A. 2001. The role 300: 1542–1548. of the Mre11–Rad50–Xrs2 complex in telomerase-mediated lengthening of Saccharomyces cerevisiae telomeres. Curr. Biol. 11: 1328–1335. Ueno, M., Nakazaki, T., Akamatsu, Y., Watanabe, K., Tomita, K., Lindsay, H.D., Shinagawa, H., and Iwasaki, H. 2003. Mo- lecular characterization of the Schizosaccharomyces pombe nbs1+ gene involved in DNA repair and telomere mainte- nance. Mol. Cell. Biol. 23: 6553–6563. Unsal-Kacmaz, K. and Sancar A. 2004. Quaternary structure of ATR and effects of ATRIP and replication protein A on its DNA binding and kinase activities. Mol. Cell. Biol. 24: 1292–1300. Usui, T., Ogawa, H., and Petrini, J.H.J. 2001. A DNA damage response pathway controlled by Tel1 and the Mre11 com- plex. Mol. Cell 7: 1255–1266. Uziel, T., Lerenthal, Y., Moyal, L., Andegeko, Y., Mittelman, L., and Shiloh, Y. 2003. Requirement of the MRN complex for ATM activation by DNA damage. Embo J. 22: 5612–5621. van Steensel, B., Smogorzewska, A., and de Lange, T. 1998. TRF2 protects human telomeres from end-to-end fusions. Cell 92: 401–413. Varley, H., Pickett, H.A., Foxon, J.L., Reddel, R.R., and Royle, N.J. 2002. Molecular characterization of inter-telomere and intra-telomere mutations in human ALT cells. Nat. Genet. 30: 301–305. Viscardi, V., Baroni, E., Romano, M., Lucchini, G., and Longhese, M.P. 2003. Sudden telomere lengthening triggers a Rad53- dependent checkpoint in Saccharomyces cerevisiae. Mol. Biol. Cell 14: 3126–3143. von Zglinicki, T. 2002. Oxidative stress shortens telomeres. Trends Biochem. Sci. 27: 339–344. Wei, C., Skopp, R., Takata, M., Takeda, S., and Price, C.M. 2002. Effects of double-strand break repair proteins on vertebrate telomere structure. Nucleic Acids Res. 30: 2862–2870. Weizman, N., Shiloh, Y., and Barzilai, A. 2003. Contribution of the Atm protein to maintaining cellular homeostasis evi- denced by continuous activation of the AP-1 pathway in Atm-deficient brains. J. Biol. Chem. 278: 6741–6747. Wilson, S., Warr, N., Taylor, D.L., and Watts, F.Z. 1999. The role of Schizosaccharomyces pombe Rad32, the Mre11 ho- mologue, and other DNA damage response proteins in non- homologous end joining and telomere length maintenance. Nucleic Acids Res. 27: 2655–2661. Wong, K.-K., Chang, S., Weiler, S.R., Ganesan, S., Chaudhuri, J., Zhu, C., Artandi, S.E., Rudolph, K.L., Gottlieb, G.J., Chin, L., et al. 2000. Telomere dysfunction impairs DNA repair and enhances sensitivity to ionizing radiation. Nat. Genet. 26: 85–88. Wong, J.M., Kusdra, L., and Collins, K. 2002. Subnuclear shut- tling of human telomerase induced by transformation and DNA damage. Nat. Cell. Biol. 4: 731–736. Zhou, B.-B.S. and Elledge, S.J. 2000. The DNA damage response: Putting checkpoints in perspective. Nature 408: 433–439. Zhu, X.-D., Küster, B., Mann, M., Petrini, J.H.J., and Lange, T.D. 2000. Cell-cycle-regulated association of RAD50/MRE11/ NBS1 with TRF2 and human telomeres. Nat. Genet. 25: 347–352. Zhu, X.D., Niedernhofer, L., Kuster, B., Mann, M., Hoeijmakers, J.H., and de Lange, T. 2003. ERCC1/XPF removes the 3Ј over- hang from uncapped telomeres and represses formation of telomeric DNA-containing double minute chromosomes.

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Functional links between telomeres and proteins of the DNA-damage response

Fabrizio d'Adda di Fagagna, Soo-Hwang Teo and Stephen P. Jackson

Genes Dev. 2004, 18: Access the most recent version at doi:10.1101/gad.1214504

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